阅读说明：本技术 食蚊鱼Nrf2基因及其提取方法及特异性一抗抗体制备方法 (Mosquito-eating fish Nrf2 gene, extraction method thereof and preparation method of specific primary anti-antibody ) 是由 鲍爽 聂湘平 于 2020-06-08 设计创作，主要内容包括：本发明公开了一种食蚊鱼Nrf2基因及其提取方法及特异性一抗抗体制备方法,首次成功克隆了食蚊鱼体内的Nrf2基因的核心碱基序列,并合成了食蚊鱼Nrf2特异性多克隆一抗抗体,可推广用于快速获得其他非模式物种未知碱基序列及制备相关特异性抗体,并在后续研究中使用这些基因构建分子层面检测方法和使用抗体从事相关蛋白变化的检测。食蚊鱼环境适应力超强,甚至在生物需氧量和化学需氧量较高的污水和下水道中都能正常生活,推测其超强生命力也和其体内的Nrf2抗氧化信号通路有关,本方法克隆出的基因和制备出的一抗抗体可为研究Nrf2相关通路如何在环境压力下发挥抗氧化作用、生物如何抵御氧化应激压力和相关环境风险提供新的技术手段和检测方法。(The invention discloses a mosquito-eating fish Nrf2 gene, an extraction method thereof and a preparation method of a specific primary anti-antibody, successfully clones a core base sequence of the Nrf2 gene in a mosquito-eating fish body for the first time, synthesizes the specific polyclonal primary anti-antibody of the Nrf2 gene of the mosquito-eating fish, can be popularized and used for quickly obtaining unknown base sequences of other non-model species and preparing related specific antibodies, and uses the genes to construct a molecular level detection method and uses the antibodies to carry out detection on related protein changes in subsequent researches. The mosquito-eating fish has super-strong environmental adaptability, can normally live even in sewage and sewers with higher biological oxygen demand and chemical oxygen demand, supposes that the super-strong vitality of the mosquito-eating fish is also related to an Nrf2 antioxidant signal channel in the mosquito-eating fish, and provides a new technical means and a detection method for researching how an Nrf2 related channel plays an antioxidant effect under environmental pressure, how organisms resist oxidative stress pressure and related environmental risks.)

1. A mosquito-eating fish Nrf2 gene is characterized in that the sequence of the gene fragment is shown as SEQ ID NO. 1.

2. The extraction method of the Nrf2 gene of the mosquito-eating fish as claimed in claim 1, comprising the following steps:

1) extracting total RNA of the liver of the mosquito-eating fish;

2) carrying out reverse transcription on the total RNA of the liver of the mosquito-eating fish to obtain a first chain cDNA of the mosquito-eating fish;

3) carrying out common PCR amplification by taking the first chain cDNA of the mosquito-eating fish as a template to obtain a core fragment of the mosquito-eating fish Nrf 2;

4) uniformly mixing the product subjected to PCR amplification with 5 × Loading Buffer, injecting the mixture into a sample application hole on gel, adding a Marker, and running for 40min under the voltage of 90V of an electrophoresis apparatus;

5) and detecting the electrophoresis result of the gel after electrophoresis by using a ChemiDoc XRS + gel imaging system and ultraviolet light, cutting the gel at the position of the strip which accords with the designed length, and sequencing the cut gel.

3. The extraction method of the Nrf2 gene of the mosquito-eating fish as claimed in claim 2, wherein the extraction of the total RNA of the liver of the mosquito-eating fish comprises the following steps:

1) RAN extraction: putting the liver of the mosquito-eating fish into a round bottom centrifugal tube which is subjected to sterile treatment, adding Trizol Reagent solution, sucking and blowing by using a 0 disposable sterile syringe until the solution is uniform pink red homogenate, sucking the solution by using a needle without blockage and obvious blocks; standing the uniform pink homogenate in a round bottom centrifuge tube at room temperature for 5 min; adding 200 μ L chloroform, covering the centrifugal tube, shaking the centrifugal tube, mixing, and standing at room temperature for 5 min; centrifuging the centrifuge tube at 4 deg.C and 12000g for 15 min;

2) RNA precipitation: sucking the supernatant into a sterile conical-bottom centrifuge tube, adding isopropanol, slightly turning the tube cover upside down for several times, standing at room temperature for 10min, centrifuging the conical-bottom centrifuge tube at 4 ℃ and 12000g for 10min, and forming a white gel-like precipitate at the tube bottom after centrifugation;

3) RNA purification: removing supernatant from the pointed-bottom centrifuge tube, adding 75% sterile ethanol, and flicking the bottom of the tube with fingers to suspend the precipitate; centrifuging at 4 deg.C and 7500g for 5min, and carefully sucking off the supernatant with a pipette; drying for 5-10 min at room temperature to volatilize ethanol; RNase FreeH2O was added to dissolve the RNA.

4. The method for extracting the gene of the mosquito-eating fish Nrf2 as claimed in claim 2, wherein when ordinary PCR amplification is performed to obtain the core fragment of the mosquito-eating fish Nrf2, the reaction system of ordinary PCR is ddH2O 17.25.25 μ L,10 XPCR buffer 2.5 μ L, dNTP mix 2 μ L, cDNA template 0.75 μ L, upstream and downstream primers are 1 μ L each, rTaq enzyme 0.5 μ L, the reaction system is 25 μ L, the ordinary PCR reaction is performed in ABI Veriti 96-well gradient PCR instrument, the reaction conditions are 95 ℃, and the pre-denaturation is 4 min; performing denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, extending at 72 ℃ for 1min, and circulating for 35 times; extending for 10min at 72 ℃; storing at 4 ℃.

5. The method for extracting the Nrf2 gene of the mosquito-eating fish as claimed in claim 4, wherein the primers need to select the gene sequence of the fish close to the genetic relationship with the mosquito-eating fish as reference according to the existing data of NCBI database and Ensemblosia database, search the conserved region among several species close to the genetic relationship with the mosquito-eating fish, and design the primers according to the design requirements of common PCR primers in the conserved region.

6. The method for extracting the Nrf2 gene of the mosquito-eating fish as claimed in claim 1, wherein a buffer solution and 2% agarose gel are added during electrophoresis.

7. A method for preparing a specific primary antibody of a mosquito-eating fish Nrf2 gene, which is characterized in that sensitive polypeptide chains are analyzed according to the sequence of the mosquito-eating fish Nrf2 of claim 1, and the specific polyclonal primary antibody of the mosquito-eating fish Nrf2 protein is synthesized.

8. The method for preparing the specific primary antibody of the Nrf2 gene of the mosquito-eating fish as claimed in claim 7, which comprises the following steps:

1) respectively synthesizing NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide corresponding to 41 th to 52 th bases;

2) respectively carrying out KLH coupling on the synthesized NRF2 polypeptide corresponding to 606 th to 617 th bases and the synthesized NRF2 polypeptide corresponding to 41 th to 52 th bases, and mixed immunizing 2 New Zealand white rabbits for 400 mu g/time of subcutaneous immunization, one immunization for 2-3 weeks and 4 times of immunization;

3) performing antiserum titer detection;

4) and (5) purifying and identifying the antibody.

9. The method for preparing the specific primary antibody of the Nrf2 gene of the mosquito-eating fish as claimed in claim 8, wherein the step of detecting the indirect titer of the antiserum comprises the following steps:

1) designing a coating plate according to the experimental requirement, and marking the lath;

2) coating: diluting NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide antigen corresponding to 41 th to 52 th bases to required concentration by PBS coating liquid, mixing uniformly, adding into laths, each hole being 100ul, and refrigerating at 4 ℃ overnight;

coating antigen: NRF2 polypeptide corresponding to bases 606 to 617, NRF2 polypeptide corresponding to bases 41 to 52;

coating concentration: 5 mu g/ml,100 ul/well;

coating buffer solution: phosphate buffer;

3) and (3) sealing: after coating, removing coating liquid, washing the plate for 3 times, adding 200 mu L of sealing liquid into each hole, keeping the temperature constant for 1h at 37 ℃, taking out the enzyme label plate, removing the inner liquid, and washing the plate for 1 time;

4) primary anti-reaction: diluting antiserum by 1/500, 3 times, wherein each well is 100 μ L, and incubating at 37 deg.C for 1 h;

5) secondary antibody reaction: taking out the enzyme label plate, discarding the internal liquid, washing the plate for 3 times, adding 100 mu L of diluted enzyme-labeled secondary antibody and enzyme-labeled secondary antibody into each hole: goat anti-rabbit-HRP, 1/5000, thermostat at 37 ℃ for 1 hour;

6) color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ L of TMB color development solution into each hole, determining the color development time according to the color depth, generally 37 deg.C, 15 min;

7) and (3) terminating the reaction: add 100. mu.L of 1M HCl solution to each well, terminate the reaction, immediately read 450nm on the microplate reader, and determine the titer of the sample by the dilution corresponding to the well whose OD value is greater than 2.1 times the set negative control OD value.

10. The method for preparing the mosquito-eating fish Nrf2 gene specific primary antibody according to claim 8, wherein the antibody purification comprises the following steps:

preparing sulfhydryl glue, NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide mixed polypeptide corresponding to 41 th to 52 th bases into an antigen affinity purification chromatographic column, mixing rabbit antiserum and PBS in equal amount, slowly loading the mixture, eluting the mixture with glycine elution buffer solution after the antibody is combined to obtain the required purified antibody, dialyzing the mixture in the PBS at 4 ℃ overnight immediately, and measuring the purity, concentration and titer every other day;

the identification of the antibody comprises the following steps:

detecting the titer of the purified antibody by ELISA, and performing concentration determination on the obtained antibody by using a BCA protein concentration determination kit; the purity of the purified antibody was observed by SDS-PAGE, staining with Coomassie Brilliant blue.

Technical Field

The invention relates to the technical field of gene extraction and specific antibody preparation, in particular to a mosquito-eating fish Nrf2 gene, an extraction method thereof and a preparation method of a specific anti-antibody.

Background

Mosquito-eating fish (Gambusia affinis) is a small freshwater fish, native to south America, belonging to the genus Gambusia of family medakaceae. The female fish and the male fish are obviously different in body types: the female fish is generally larger than the male fish in body shape, the hip fins of the female fish are fan-shaped, and the hip fins of the male fish are sword-shaped. The mosquito-eating fish is also called big belly fish and willow fish, and the breeding mode is egg birth. Mosquito-eating fish is widely introduced to control mosquito-related epidemic situation in various places around the world due to its strong environmental adaptability and broad-spectrum feeding property. In recent years, mosquito-eating fishes have been widely present in river water in china as an invasive species. The mosquito-eating fish is suitable for being used as an experimental animal in the aspect of ecological environment research due to obvious sex difference, wide distribution in natural water areas, easy capture and easy domestication in laboratories. At present, some researches and reports have been made on mosquito-eating fish as experimental animals. However, the mosquito-eating fish is not a rare protective species, and even belongs to invasive species for China, the mosquito-eating fish has strong environmental adaptability and fertility, even occupies living spaces of a plurality of species (such as medaka) with similar local ecological niches in China, and most researchers still remain on the superficial level for research. In addition, the genetic background of mosquito-eating fish is unclear, and the sequences of the complete genome thereof are not included in the NCBI database. The related researches mostly focus on the aspects of apparent character change, population structure and the like of the mosquito-eating fish, and the related researches and methods on the molecular biology level of the gene are rarely discussed.

The Nrf2 nuclear transcription factor (NF-E2-related factor 2), also known as NFE2L2, is a nuclear transcription factor containing a basic leucine zipper structure. Nrf2 is a very important and evolutionarily conserved transcription factor for regulating organism oxidative stress response, and generally regulates the expression of downstream antioxidant related genes and II phase detoxification metabolic enzyme genes through an Nrf2/ARE (antioxidant response element) signal channel so as to maintain and protect the balance of the redox state in the organism.

In view of this, there is an urgent need to improve the conventional gene extraction method to facilitate the operation and obtain the Nrf2 gene and specific primary antibody of mosquito-eating fish.

Disclosure of Invention

The invention aims to solve the technical problems that the genetic background of the mosquito-eating fish is not clear, and the NCBI database does not record the sequence of the whole genome of the mosquito-eating fish.

In order to solve the technical problems, the invention adopts the technical scheme that the mosquito-eating fish Nrf2 gene is characterized in that the sequence of the gene fragment is shown as Nrf2 ID NO. 1.

The invention adopts another technical scheme that the method for extracting the Nrf2 gene of the mosquito-eating fish comprises the following steps:

1) extracting total RNA of the liver of the mosquito-eating fish;

2) carrying out reverse transcription on the total RNA of the liver of the mosquito-eating fish to obtain a first chain cDNA of the mosquito-eating fish;

3) carrying out common PCR amplification by taking the first chain cDNA of the mosquito-eating fish as a template to obtain a core fragment of the mosquito-eating fish Nrf 2;

4) uniformly mixing the product after completing the PCR amplification with 5 × Loading Buffer, injecting into a sample application hole on the gel, adding a proper Marker, and running for 40min under the voltage of 90V of an electrophoresis apparatus;

5) and detecting the electrophoresis result of the gel after electrophoresis by using a ChemiDoc XRS + gel imaging system and ultraviolet light, cutting the gel at the position of the strip which accords with the designed length, and sequencing.

In the above technical scheme, the extracting total RNA from the liver of the mosquito-eating fish comprises:

1) RAN extraction: putting the liver of the mosquito-eating fish into a round bottom centrifugal tube which is subjected to sterile treatment, adding TrizolReagent solution, sucking and blowing by using a 0 disposable sterile syringe until the solution is uniform pink homogenate, and sucking the solution by using a needle without blockage or obvious blocks; standing the uniform pink homogenate in a round bottom centrifuge tube at room temperature for 5 min; adding 200 μ L chloroform, covering the centrifugal tube, shaking the centrifugal tube, mixing, and standing at room temperature for 5 min; centrifuging the centrifuge tube at 4 deg.C and 12000g for 15 min;

2) RNA precipitation: sucking the supernatant into a sterile conical-bottom centrifuge tube, adding isopropanol, slightly turning the tube cover upside down for several times, standing at room temperature for 10min, centrifuging the conical-bottom centrifuge tube at 4 ℃ and 12000g for 10min, and forming a white gel-like precipitate at the tube bottom after centrifugation;

3) RNA purification: removing supernatant from the pointed-bottom centrifuge tube, adding 75% sterile ethanol, and flicking the bottom of the tube with fingers to suspend the precipitate; centrifuging at 4 deg.C and 7500g for 5min, and carefully sucking off the supernatant with a pipette gun; drying for 5-10 min at room temperature to volatilize ethanol; RNase Free H2O was added to dissolve the RNA.

In the technical scheme, when common PCR amplification is carried out to obtain the core fragment of the mosquito-eating fish Nrf2, the reaction system of the common PCR is ddH2O 17.25.25 muL, 10 XPCR Buffer 2.5 muL, dNTP mix 2 muL, cDNA template 0.75 muL, upstream and downstream primers (10 muM concentration) 1 muL respectively, rTaq enzyme 0.5 muL, and the total reaction system is 25 muL. Carrying out common PCR reaction in ABIVeriti 96-hole gradient PCR instrument under the reaction condition of 95 ℃ and carrying out pre-denaturation for 4 min; performing denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, extending at 72 ℃ for 1min, and circulating for 35 times; extending for 10min at 72 ℃; storing at 4 ℃.

In the technical scheme, the primers need to select the gene sequence of the fish close to the genetic relationship with the mosquito-eating fish as reference according to the existing data of an NCBI database and an Ensemblosia database, search conserved regions among several species close to the genetic relationship with the mosquito-eating fish, and design the primers in the conserved regions according to the design requirements of common PCR primers.

In the above technical scheme, a buffer solution and 2% agarose gel are required to be added during electrophoresis.

The other technical scheme provided by the invention is a preparation method of the mosquito-eating fish Nrf2 gene specificity primary antibody, which comprises the steps of analyzing sensitive polypeptide chains according to the sequence of the mosquito-eating fish Nrf2, and synthesizing the mosquito-eating fish Nrf2 protein specificity polyclonal primary antibody.

In the technical scheme, the preparation method of the specific primary anti-antibody of the mosquito-eating fish Nrf2 gene specifically comprises the following steps:

1) respectively synthesizing NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide corresponding to 41 th to 52 th bases;

2) respectively carrying out KLH coupling on the synthesized NRF2 polypeptide corresponding to 606 th to 617 th bases and the synthesized NRF2 polypeptide corresponding to 41 th to 52 th bases, and then carrying out mixed immunization on 2 New Zealand white rabbits for subcutaneous immunization at 400 mu g/time, one immunization at 2-3 weeks and 4 times of immunization;

3) performing antiserum titer detection;

4) and (5) purifying and identifying the antibody.

In the technical scheme, the antiserum indirect titer detection method specifically comprises the following steps:

1) designing a coating plate according to the experimental requirement, and marking the lath;

2) coating: diluting NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide antigen corresponding to 41 th to 52 th bases to required concentration by PBS coating liquid, mixing uniformly, adding into laths, each hole being 100ul, and refrigerating at 4 ℃ overnight;

coating antigen: NRF2 polypeptide corresponding to bases 606 to 617, NRF2 polypeptide corresponding to bases 41 to 52;

coating concentration: 5 mu g/ml,100 ul/well;

coating buffer solution: phosphate buffer;

3) and (3) sealing: after coating, removing coating liquid, washing the plate for 3 times, adding 200 mu L of sealing liquid into each hole, keeping the temperature constant for 1h at 37 ℃, taking out the enzyme label plate, removing the inner liquid, and washing the plate for 1 time;

4) primary anti-reaction: diluting antiserum by 1/500, 3 times, wherein each well is 100 μ L, and keeping the temperature at 37 ℃ for 1 h;

5) secondary antibody reaction: taking out the enzyme label plate, discarding the internal liquid, washing the plate for 3 times, adding 100 mu L of diluted enzyme-labeled secondary antibody and enzyme-labeled secondary antibody into each hole: goat anti-rabbit-HRP, 1/5000, thermostat at 37 ℃ for 1 hour;

6) color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ L of TMB color development solution into each hole, determining the color development time according to the color depth, generally 37 deg.C, 15 min;

7) and (3) terminating the reaction: the reaction was stopped by adding 100. mu.L of 1M HCl solution to each well, immediately reading the wells at 450nm on an enzyme calibrator, and determining the titer of the sample as the dilution corresponding to the well with an OD value greater than 2.1 times the OD value of the negative control set.

In the above technical scheme, the purification of the antibody comprises the following steps:

preparing sulfhydryl glue, NRF2 polypeptide corresponding to 606 th to 617 th bases and NRF2 polypeptide mixed polypeptide corresponding to 41 th to 52 th bases into an antigen affinity purification chromatographic column, mixing rabbit antiserum and PBS in equal amount, slowly loading the mixture, after the antibody is combined, respectively eluting the mixture by using glycine elution buffer solution to obtain the required purified antibody, immediately dialyzing the mixture in the PBS at 4 ℃ overnight, and measuring the purity, concentration and titer every other day;

the identification of the antibody comprises the following steps:

detecting the titer of the purified antibody by ELISA, and performing concentration determination on the obtained antibody by using a BCA protein concentration determination kit; the purity of the purified antibody was observed by SDS-PAGE electrophoresis, staining with Coomassie Brilliant blue.

Compared with the prior art, the extraction method of the mosquito-eating fish Nrf2 gene and the preparation method of the specific primary anti-antibody successfully clone the core base sequence of the Nrf2 gene in the mosquito-eating fish for the first time, synthesize the specific polyclonal primary anti-antibody of the mosquito-eating fish Nrf2, can be popularized and used for rapidly obtaining unknown base sequences of other non-model species and preparing related specific antibodies, and use the genes to construct a molecular level detection method and use the antibodies to perform detection on related protein changes in subsequent researches. The mosquito-eating fish has super strong environmental adaptability, is a strong invasive organism in China, can normally live even in sewage and sewers with higher biological oxygen demand and chemical oxygen demand, and is conjectured that the super strong vitality is also related to the in-vivo Nrf2 antioxidant signal pathway, and the gene cloned by the method and the prepared primary antibody can provide a new technical means and a detection method for researching how the Nrf2 related pathway plays an antioxidant role under environmental pressure, how organisms resist oxidative stress pressure and related environmental risks.

Drawings

FIG. 1 shows the result of gel electrophoresis of the mosquito-eating fish Nrf2 gene;

FIG. 2 is an Nrf2 gene evolutionary tree of a mosquito-eating fish of the present invention;

FIG. 3 shows the HPLC experimental results of the polypeptide NRF2(606-617) of the present invention;

FIG. 4 shows the result of MS experiment with polypeptide NRF2(606-617) of the present invention;

FIG. 5 shows the HPLC experimental results of NRF2(41-52) polypeptide of the present invention;

FIG. 6 shows the result of MS experiment of polypeptide NRF2(41-52) of the present invention;

FIG. 7 shows the SDS-PAGE result of polyclonal antibody against Nrf2 of mosquito-eating fish in the present invention;

FIG. 8 shows the experimental results of a polyclonal primary anti-antibody WB assay of mosquito-eating fish Nrf2 according to the present invention.

Detailed Description

The invention provides a method for extracting mosquito-eating fish Nrf2 gene and a method for preparing specific primary anti-antibody, which are convenient to operate and can obtain mosquito-eating fish Nrf2 gene and specific primary anti-antibody. The invention is described in detail below with reference to the drawings and the detailed description.

The mosquito-eating fish Nrf2 gene is characterized in that the sequence of the gene fragment is shown as Nrf2 IDNO.1.

The invention provides a method for extracting mosquito-eating fish Nrf2 gene, which is characterized by comprising the following steps:

1) extracting total RNA of the liver of the mosquito-eating fish;

2) carrying out reverse transcription on the total RNA of the liver of the mosquito-eating fish to obtain a first chain cDNA of the mosquito-eating fish;

3) carrying out common PCR amplification by taking the first chain cDNA of the mosquito-eating fish as a template to obtain a core fragment of the mosquito-eating fish Nrf 2;

4) uniformly mixing the product after completing the PCR amplification with 5 × Loading Buffer, injecting into a sample application hole on the gel, adding a proper Marker, and running for 40min under the voltage of 90V of an electrophoresis apparatus;

5) and detecting the electrophoresis result of the gel after electrophoresis by using a ChemiDoc XRS + gel imaging system and ultraviolet light, cutting the gel at the position of the strip which accords with the designed length, and sequencing.

In the embodiment, a first chain cDNA of the mosquito-eating fish is obtained by reverse transcription of RNA of the mosquito-eating fish, a core base sequence of an Nrf2 gene in the mosquito-eating fish is successfully cloned by means of technical means such as PCR amplification, the genetic characteristics of the Nrf2 gene in the mosquito-eating fish can be analyzed according to a central sequence of an Nrf2 gene in the mosquito-eating fish, an Nrf2 evolutionary tree is manufactured to compare the evolutionary positions of the Nrf2 gene, the whole-gene sequencing of the mosquito-eating fish is not needed, a core fragment of the Nrf2 gene in the mosquito-eating fish is obtained, and the method is simple and easy to operate, and can be popularized to the acquisition of gene fragments of.

In one embodiment of the present invention, preferably, extracting mosquito-eating fish liver total RNA comprises:

1) RAN extraction: quickly placing the livers of the mosquitos dissected from the ice tray into a sterile-processed 2mL round-bottom centrifugal tube, adding 1mL Trizol Reagent solution, sucking and blowing by a disposable sterile syringe with the volume of 1mL and a 0.45-micrometer opening needle head until the solution is uniform pink homogenate, and sucking the solution by the needle head without blockage and obvious blocks; standing the 2mL round-bottom centrifuge tube for 5min at room temperature; adding 200 μ L chloroform, covering the centrifugal tube, shaking the centrifugal tube, and standing at room temperature for 5 min; centrifuging 2mL round-bottom centrifuge tubes at 4 ℃ and 12000g for 15 min; after centrifugation, the mixture was divided into three layers, wherein RNA was present in the upper colorless aqueous phase;

2) RNA precipitation: carefully sucking the supernatant into a 1.5mL sterile conical centrifuge tube, adding 500. mu.L isopropanol, covering the tube cover, slightly turning upside down for several times, and standing at room temperature for 10 min; centrifuging a 1.5mL conical-bottom centrifuge tube at 4 ℃ and 12000g for 10min, and forming white gel sample precipitation at the tube bottom after centrifugation; (ii) a

3) RNA purification: 1.5mL of a conical-bottomed centrifuge tube, discarding the supernatant, adding 1mL of 75% sterile processing ethanol, and flicking the tube bottom with fingers to suspend the precipitate; 1.5mL of a conical centrifuge tube was centrifuged at 7500g for 5min at 4 ℃ and the supernatant carefully aspirated off with a pipette; drying at room temperature for 5-10 min to volatilize ethanol (but care is not taken to completely dry the RNA, and the RNA is difficult to dissolve if the RNA is completely dried); add 20-30. mu.L RNase Free H2O to dissolve the RNA.

4) RNA detection: the extracted total RNA of the liver of the mosquito-eating fish is detected by using a Nano Drop 2000 ultramicro spectrophotometer to detect the extraction quality, the absorbance OD 260/280 of the total RNA meets the requirement of 1.8-2.0, and the successfully extracted RNA is temporarily stored in an ultra-low temperature refrigerator at minus 80 ℃.

Wherein, the above experiment should be carried out under superclean bench or aseptic environment in the whole journey, and all centrifuging tubes and rifle head that use all are through not having the enzymatic treatment.

The first strand cDNA of the mosquito-eating fish is obtained by reverse transcription of total liver RNA, the operation follows the instruction of a PrimeScriptTM II RT reagent Kit of TaKaRa company, the whole experiment is carried out under an ultraclean workbench by wearing sterile latex gloves, the operation on ice is kept, all centrifuge tubes and gun heads are subjected to non-enzymatic treatment, and the reverse transcription reaction system is shown in table 1.

TABLE 1 reverse transcription reaction System for first Strand cDNA Synthesis

In one embodiment of the present invention, preferably, when performing ordinary PCR amplification to obtain core fragment of Nrf2 of mosquito-eating fish, the reaction system of ordinary PCR is ddH2O 17.25.25. mu.L, 10 XPCR Buffer 2.5. mu.L, dNTP mix 2. mu.L, cDNA template 0.75. mu.L, upstream and downstream primers (10. mu.M concentration) 1. mu.L each, rTaq enzyme 0.5. mu.L, and the reaction system is 25. mu.L. Carrying out common PCR reaction in ABI Veriti 96-hole gradient PCR instrument under the reaction condition of 95 ℃ and carrying out pre-denaturation for 4 min; performing denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, extending at 72 ℃ for 1min, and circulating for 35 times; extending for 10min at 72 ℃; storing at 4 ℃.

In one embodiment of the invention, preferably, the primers need to select the gene sequence of the fish close to the relation of the mosquito-eating fish as reference according to the existing data of the NCBI database and the Ensemblosia database, search conserved regions among several species close to the relation of the mosquito-eating fish, and design the primers in the conserved regions according to the design requirements of common PCR primers.

In this example, based on the existing data of the NCBI database and EnsemblASIA database, fishes of the order medaka that are relatively close to the relativity of mosquito-eating fishes (e.g., Camphora, medaka, etc.) can be selected with reference to the gene sequences of other bony fishes (e.g., typical model species zebrafish and rainbow trout), conserved regions among the above species are searched on Vector NTI 11.5.1 software, and primers are designed in the conserved regions according to the general PCR primer design requirements, as shown in Table 2. The design of common PCR primers of the mosquito-eating fish Nrf2 refers to the following gene sequences respectively: nrf2 primers were designed from Nrf2 sequences numbered HQ916348.1 (rainbow trout, Oncorhynchus mykiss), XM _005172511.1 (zebrafish, Danio rerio) and ENSXMAT00000000447 (Xiphophorus macultus, obtained from the Ensembl ASIA database).

The designed common PCR primers are diluted to 10 mu M concentration by RNase Free dH2O, and are separately stored in a low-temperature refrigerator at-20 ℃ to avoid repeated freeze thawing as much as possible.

TABLE 2 primer design for mosquito-eating fish Nrf2

F, upstream; r is downstream.

In one embodiment of the present invention, it is preferable to add buffer and 2% agarose gel when performing electrophoresis.

In this embodiment, the buffer solution is prepared by the following method: 242g of Tris (hydroxymethyl) aminomethane (Tris) and EDTA18.612g are weighed into a 1L beaker; adding about 800mL of deionized water into a beaker, and fully and uniformly stirring; adding 57.1mL of glacial acetic acid, and fully dissolving; adjusting the pH value to 8.3 by NaOH, then fixing the volume to 1L by deionized water, and storing at room temperature. When in use, the mixture is diluted by 50 times by deionized water to obtain 1 × TAE Buffer. The normal PCR product was electrophoresed in 2% agarose gel to run clear bands and the results were examined. The preparation method of the 2% agarose gel comprises the following steps: 0.36g of agarose gel powder is added into 18mL of 1 XTAE Buffer, the mixture is heated by microwave to be melted to be colorless and transparent, 200 mu LGel Green dye (diluted according to the ratio of 1: 10000) is added after the mixture is cooled slightly and poured into a template for casting, and the mixture is cooled at room temperature for 40min to obtain the 2 percent agarose gel.

According to the invention, an Nrf2 sequence with the length of 803bp is successfully cloned, the electrophoresis picture of the sequence is shown in figure 1, and the longest Open Reading Frame (ORF) of the sequence is 758bp and can be transcribed into 252 amino acids which are analyzed in Vector NTI software and NCBI database, and the predicted protein molecular weight of the polypeptide chain is 28,757kDa, and the theoretical pI value is 4.54, as shown in a sequence list. To examine the evolutionary relationship of detoxified metabolism-related genes of mosquito-eating fishes and other species, we constructed an evolutionary tree from the base sequences of Nrf2 of different species using MEGA5.05 software, as shown in fig. 2.

A method for preparing a specific monoclonal antibody of a mosquito-eating fish Nrf2 gene comprises the steps of analyzing a sensitive polypeptide chain according to the sequence of the mosquito-eating fish Nrf2 in the embodiment, and synthesizing the specific polyclonal monoclonal antibody of the mosquito-eating fish Nrf2 protein.

In the embodiment, the mosquito-eating fish Nrf2 specific polyclonal primary antibody is synthesized according to the core base sequence of the Nrf2 gene of the mosquito-eating fish body, can be popularized and used for quickly obtaining unknown base sequences of other non-model species and preparing related specific antibodies, and can be used for constructing a molecular level detection method and detecting related protein changes by using the genes in subsequent researches.

In one embodiment of the present invention, preferably, the method specifically includes:

1) separately synthesizing a synthetic NRF2(606-617) polypeptide and a NRF2(41-52) polypeptide;

2) after KLH coupling of the synthesized polypeptides NRF2(606-617) and NRF2(41-52), 2 New Zealand white rabbits (2-2.5kg) were mixed immunized with 400. mu.g/time subcutaneous once for 2-3 weeks and 4 times of immunization

3) Performing antiserum titer detection;

4) and (5) purifying and identifying the antibody.

In this example, NRF2(606-617) polypeptides were synthesized, and specific HPLC and MS reports are shown in FIG. 3, FIG. 4, and NRF2(41-52) polypeptides, and specific HPLC and MS reports are shown in FIG. 5, FIG. 6.

In one embodiment of the present invention, preferably, the step of detecting the indirect titer of the antiserum is specifically as follows:

1) designing a coating plate according to the experimental requirements, and marking the plate strip

2) Coating: the polypeptides NRF2(606-617) and NRF2(41-52) antigens were diluted to the required concentrations with PBS coating solution, mixed well and added to the strips at 100ul per well overnight in a refrigerator at 4 ℃.

Coating antigen: polypeptide NRF2(606-617), polypeptide NRF2 (41-52);

coating concentration: 5 mu g/ml,100 ul/well;

coating buffer solution: phosphate buffered saline (PBS, pH 7.4).

3) And (3) sealing: after coating, the coating solution was discarded, the plate was washed 3 times, 200. mu.L of the blocking solution was added to each well, and the plate was incubated at 37 ℃ for 1 hour. The ELISA plate was removed, the internal solution was discarded, and the plate was washed 1 time.

4) Primary anti-reaction: the antiserum was diluted 1/500, 3-fold, 100. mu.L per well, incubated at 37 ℃ for 1 h.

5) Secondary antibody reaction: taking out the enzyme label plate, discarding the internal liquid, washing the plate for 3 times, adding 100 mu L of diluted enzyme-labeled secondary antibody and enzyme-labeled secondary antibody into each hole: goat anti-rabbit-HRP, 1/5000. Incubator 1 hour at 37 ℃.

6) Color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ L of TMB color developing solution into each hole, determining the color developing time according to the color depth, generally 37 deg.C, 15 min.

7) And (3) terminating the reaction: the reaction was stopped by adding 100. mu.L of 1M HCl solution to each well. The titer of the sample is determined by reading immediately on an enzyme calibrator at 450nm and diluting the wells with an OD 2.1 times greater than the OD of the negative control set.

In this example, the antiserum titer was greater than 121K, with specific results as shown in table 3, coating the antigen: polypeptide NRF2(606-617), polypeptide NRF2(41-52), coating concentration: 5 μ g/ml,100 μ L/well, coating buffer: phosphate buffered saline (PBS, pH7.4), secondary antibody: goat anti-rabbit-HRP, 1/5000.

TABLE 3 antiserum Elisa results

Initial dilution 1:500

In one embodiment of the present invention, preferably, performing purification of the antibody comprises: preparing the sulfhydryl glue, mixed polypeptide of NRF2(606-617) and NRF2(41-52) into an antigen affinity purification chromatographic column, mixing rabbit antiserum and PBS in equal amount, slowly loading the rabbit antiserum, combining the antibodies, then respectively eluting the rabbit antiserum and the PBS by using glycine elution buffer solution to obtain the required purified antibody, immediately dialyzing the antibody in the PBS at 4 ℃ overnight, and measuring the purity, the concentration and the titer every other day.

In one embodiment of the present invention, preferably, performing the identification of the antibody comprises: detecting the titer of the purified antibody by ELISA, and performing concentration determination on the obtained antibody by using a BCA protein concentration determination kit; the purity of the purified antibody was observed by SDS-PAGE, staining with Coomassie Brilliant blue.

The indirect ELISA titer detection of the purified antibody comprises the following specific operations:

(1) designing a coating plate according to the experimental requirements, and marking the plate strip

(2) Coating: the polypeptides NRF2(606-617) and NRF2(41-52) antigens are diluted to the required concentration by PBS coating liquid, mixed evenly and added into the lath, each hole is 100 mu L, and the temperature is kept in a refrigerator at 4 ℃ overnight.

Coating antigen: polypeptide NRF2(606-617), polypeptide NRF2(41-52)

Coating concentration: 5. mu.g/ml, 100. mu.L/well

Coating buffer solution: phosphate buffer (PBS, pH7.4)

(3) And (3) sealing: after coating, the coating solution was discarded, the plate was washed 3 times, 200. mu.L of the blocking solution was added to each well, and the plate was incubated at 37 ℃ for 1 hour. Taking out the ELISA plate, discarding the internal solution, and washing the plate for 1 time

(4) Primary anti-reaction: the purified antibody was diluted 2-fold at 1/500, 100. mu.L per well, in a 37 ℃ incubator for 1 h.

(5) Secondary antibody reaction: taking out the enzyme-labeled plate, discarding the internal liquid, washing the plate for 3 times, adding 100ul of diluted enzyme-labeled secondary antibody into each hole, and adding the enzyme-labeled secondary antibody: goat anti-rabbit-HRP, 1/5000. An incubator at 37 ℃ for 1 hour.

(6) Color development: taking out the enzyme label plate, discarding the inner liquid, washing the plate for 4 times, adding 100 μ L of TMB color developing solution into each hole, determining the color developing time according to the color depth, generally 37 deg.C, 15 min.

(7) And (3) terminating the reaction: the reaction was stopped by adding 100. mu.L of 1M HCl solution to each well. Immediately reading on a microplate reader at 450nm, and determining the titer of the sample according to the dilution corresponding to the well with the OD value being greater than 2.1 times of the set negative control OD value.

The purified antibody titer is greater than 512K, and the specific results are shown in the following table 2.4, and the antigen is coated: polypeptide NRF2(606-617), polypeptide NRF2(41-52), coating concentration: 5 μ g/mL,100 μ L/well, coating buffer: phosphate buffer (PBS, ph7.4), secondary antibody: goat anti-rabbit-HRP, 1/5000.

Table 4 purified antibody Elisa results

Initial dilution 1:500

Titer, i.e.the highest dilution at which sample OD/blank OD > is 2.1

When the purity of the antibody is determined, the purified antibody is subjected to SDS-PAGE and stained with Coomassie Brilliant blue. The purity of the purified antibody was found to be above 90% (FIG. 7).

After the synthesis of the primary antibody is successful, healthy mosquito-eating fish is dissected on ice, the liver of the mosquito-eating fish is taken out, and Western Blotting preliminary experiments are carried out by using the synthesized mosquito-eating fish Nrf2 protein specific primary antibody, which comprises the following specific operations:

step one, extracting total protein of liver tissues of the mosquito-eating fish: using Biyunnan RIPA (radio Immunoprecipitation assay) cell lysate containing PMSF (phenylmethylsulfonyl fluoride, protease inhibitor), adding the PMSF to the RIPA when avoiding repeated freeze thawing, and adding 99 parts of RIPA to 1 part of PMSF (PMSF final concentration is 1 mM); weighing the mass of liver tissue, adding 10 μ L lysis solution to each 10mg sample for lysis, using a glass homogenizer for full lysis, centrifuging at 14000g and 4 ℃ for 5min, taking supernatant, adding 5 × loading buffer, and subpackaging at-20 ℃ for short-term storage or storing at-80 ℃ for long-term storage without adding buffer.

Step two, detecting the total concentration of the tissue protein: the BCA protein concentration determination kit is used, and the mosquito-eating fish tissue protein concentration is detected according to the operation of the instruction. The protein sample is diluted to 10 μ g/μ L with physiological saline when adding the sample buffer solution to make the protein concentration uniform, so that the sample is conveniently loaded, and then boiled for 5min to denature the protein.

Step three, preparing protein electrophoresis colloid: the reagent kit is prepared by SDS-PAGE gel, one gel comprises 5mL of separation gel and 1.5-2mL of concentrated/stacked gel, and the specific concentration of the separation gel refers to the specification. Firstly, separating gel is prepared (the concentration of the separating gel is related to the molecular weight KDa of target protein, the concentration of the separating gel is 12% in the experiment), then the gel is concentrated (the concentration of the separating gel is 5%), APS (ammonium persulfate) and TEMED (tetramethylethylenediamine) in the gel kit are catalysts, and finally, the more TEMED is added, the faster the gel is solidified (1-2 mu L). After a separation gel solution (note: 1M Tris buffer, pH 8.8) was prepared, the gel was applied to the glass slide with a tip, and the separation gel preparation time was about 1 hour. After the separation gel is solidified, the gel is separated from water, and the upper water is poured out. Preparing concentrated gel, adding TEMED, concentrating gel liquid (injecting: using 1M Tris buffer solution, pH being 6.8), rapidly adding 1-2mL concentrated acid gel, inserting comb, preserving the gel with plastic film and double distilled water for 40min, and standing at 4 deg.C overnight. There are no bubbles in the sample well, and if there are bubbles, the well is discarded.

Step four, protein electrophoresis: after the colloid stays in the refrigerator overnight, the protein runs for electrophoresis, the comb is obliquely pulled out, the operation is careful, the glue is not damaged, and the short glass plate is moved upwards when the glue is filled into the electrophoresis tank, so that the leakage of the side surface is prevented. If 10 lanes exist, the left and right most run 1 Xprotein loading buffer solution is subjected to blank sealing, a protein molecular weight standard marker hole is reserved, a sample hole mark is made, which is known, the sample loading volume is accurate, and the total protein is consistent. When the sample is added, the electrophoresis buffer is only added into the groove, and after the sample is added, the electrophoresis buffer (the P0014BSDS-PAGE electrophoresis liquid in the cloudy day) is added to cover the potential line and add the electrode cover, so that the electrophoresis buffer cannot be reversed. The concentrated/deposited gel was run at a current of 20mA to the middle white beam portion of the gel to compress the protein into a band. The voltage of the running separation glue is as large as possible, and the marker can be stopped when the marker runs away.

Step five, film transferring: this experiment adopts the wet process to change the membrane, adopts the ice bath environment, opens the ice machine in advance, and the membrane is got to whole clamp with clean tweezers. Before membrane transfer, a PVDF (polyvinylidene fluoride) membrane is cut and activated, the PVDF membrane is immersed in methanol in a plastic box for about 1min, and the PVDF membrane is placed in a1 multiplied membrane buffer (Biyunyan day P0021BWester membrane transfer liquid) after membrane activation. The filter paper is cut out first and slightly larger than the glue, and is soaked in 1 × rotating membrane buffer. And (3) cutting corners of the PVDF film, separating the front side and the back side, keeping the glue moist during mould rotation, and sprinkling water at intervals to ensure that no air bubbles exist between the glue and the film. Making a sandwich with a rotating film: filter paper (three sheets) -gel-PVDF membrane-filter paper (three sheets) were closed vigorously and the sandwich was left unmoved with membrane and gel. The sandwich splint is put into the film transferring groove and is black, which is opposite to the black of the film transferring groove and is not needed to be reversed. Film transferring conditions: 120mA, 30-40min, (the larger the molecular weight of the target protein is, the longer the membrane conversion time is, particularly, the condition needs to be found out in advance), placing an ice bag in a groove, burying the whole groove in ice, and filling the Western membrane conversion solution in the groove.

Step six, sealing: after the membrane transfer was completed, the membrane was removed with forceps, placed in an incubation box, the shaker was opened, and the membrane was washed 3 times with TBST solution (Tris-HCl buffer + 1% volume Tween 20) for 30s each time and blocked with 5% skimmed milk powder blocking solution (Biyuntian P0023B Western blocking solution) for 2 h. The blocking was terminated and the membrane was washed 3 more times with TBST.

Step seven, incubating primary antibody: different antibodies are used for different incubation boxes, the wet filter paper is laid on the membrane during membrane shearing, the front side and the back side are clearly separated, the whole membrane is kept wet, the membrane is sheared together with required marker fragments, and the different antibodies are strictly separated. The primary antibody was kept at-20 ℃ at ordinary times and diluted 1000-fold with 5% skim milk powder blocking solution. First, the primary antibody is spotted on the incubation box, about 400 mu L of the primary antibody is used at one time, the protein surface of the membrane is contacted with the antibody, and air bubbles cannot exist between the protein surface and the antibody. Incubate the box refrigerator 4 degrees overnight.

Step eight, incubating secondary antibody: after incubation of the primary antibody, the membrane was washed with TBST and incubated with a secondary antibody, horseradish peroxidase-conjugated (primary and secondary are matched exactly) for this experiment. Secondary antibodies were also diluted with 5% milk powder TBST. The secondary antibody was incubated with the primary antibody, except that the secondary antibody was incubated at room temperature for 2 h.

Step nine, developing: chemiluminescence detection was performed using a picnic day P0018 BeyoECL Plus (hypersensitive ECL chemiluminescence kit) with reference to the instructions and results were examined using a ChemiDoc XRS + gel imaging system.

The experimental results are shown in the following fig. 8, and WB experimental results are expected, which indicates that the prepared synthetic mosquito-eating fish Nrf2 protein specific primary antibody can be used in other related protein content detection formal experiments.

The method successfully clones the core base sequence of the Nrf2 gene in the body of the mosquito-eating fish for the first time, synthesizes the specific polyclonal primary antibody of the Nrf2 of the mosquito-eating fish, can be popularized and used for quickly obtaining unknown base sequences of other non-model species and preparing related specific antibodies, and uses the genes to construct a molecular level detection method and uses the antibodies to carry out detection on related protein changes in subsequent researches. The mosquito-eating fish has super strong environmental adaptability, is a strong invasive organism in China, can normally live even in sewage and sewers with higher biological oxygen demand and chemical oxygen demand, and is conjectured that the super strong vitality is also related to the in vivo Nrf2 antioxidant signal pathway, and the gene cloned by the method and the prepared primary antibody can provide a new technical means and a detection method for researching how the Nrf2 related pathway plays an antioxidant role under the environmental pressure and how organisms resist the oxidative stress pressure and the related environmental risks.

In the present invention, the terms "first", "second", and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance; the term "plurality" means two or more unless expressly limited otherwise. The terms "mounted," "connected," "fixed," and the like are to be construed broadly, and for example, "connected" may be a fixed connection, a removable connection, or an integral connection; "connected" may be directly connected or indirectly connected through an intermediate. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The present invention is not limited to the above-mentioned preferred embodiments, and any structural changes made under the teachings of the present invention shall be understood as falling within the protection scope of the present invention.

Sequence listing

<110> river-south university

<120> mosquito-eating fish Nrf2 gene, extraction method thereof and preparation method of specific primary anti-antibody

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>803

<212>DNA

<213> mosquito-eating fish (2 Ambystoma laterale x Ambystoma jeffersonanum)

<400>1

ccgcgtacgg gcggagcttg aggaggagaa gagctgcact tgatgcagga gcaggagaag 60

gccctgctgg cgcagctgca gctggatgag gaaacgggag agtacatacc ccgcccgcct 120

cccagcgccc ctctgcagcc gaccatcaca cccctggagg ttactcagaa tgccggcttc 180

acacaggaga caggtgacag catgtcattt gatgagtgtt tgcagctcct ggcagagacg 240

tttcccatag agcaaactcc gactcccccg gtttgcctgg atgcacctgc tgtttcagta 300

cccatgatga tgtccccaaa gcagccagct ctgtcgccag ccaccctgtc ctcaggtcag 360

ccgactccgc cacagagaat gtctcccgat ctggaggagg cttggatgga gctcttgtca 420

ctccctgagc tacagcaatg tctgaacatg caaatggaag acacgttgca gaccacaaca 480

taccctctcc cagccaatgc tgaagcacag actccaagct accctaacta tccccctgtc 540

caagctccta tttttaaccc cctaaacagc atcaacaatg tcaaaacaaa cagtctgaat 600

gttggccccg cagagtttat gaatacattg gatggctcca ttcccagctt ggctccgtca 660

aatgctattc agacagatcg aagtggacgc ccccaaccta gatacaagct tcagtggaga 720

aggttttggt ggggtgtttt tatccaacaa ctgagctgga tgagtgcagc agtcagcaca 780

accaagaaga aaatgaaaga atg 803

<210>2

<211>239

<212>PRT

<213> mosquito-eating fish (2 Ambystoma laterale x Ambystoma texanum)

<400>2

Leu Leu Ala Gln Leu Gln Leu Asp Glu Glu Thr Gly Glu Tyr Ile Pro

1 5 10 15

Arg Pro Pro Pro Ser Ala Pro Leu Gln Pro Thr Ile Thr Pro Leu Glu

20 25 30

Val Thr Gln Asn Ala Gly Phe Thr Gln Glu Thr Gly Asp Ser Met Ser

35 40 45

Phe Asp Glu Cys Leu Gln Leu Leu Ala Glu Thr Phe Pro Ile Glu Gln

50 5560

Thr Pro Thr Pro Pro Val Cys Leu Asp Ala Pro Ala Val Ser Val Pro

65 70 75 80

Met Met Met Ser Pro Lys Gln Pro Ala Leu Ser Pro Ala Thr Leu Ser

85 90 95

Ser Gly Gln Pro Thr Pro Pro Gln Arg Met Ser Pro Asp Leu Glu Glu

100 105 110

Ala Trp Met Glu Leu Leu Ser Leu Pro Glu Leu Gln Gln Cys Leu Asn

115 120 125

Met Gln Met Glu Asp Thr Leu Gln Thr Thr Thr Tyr Pro Leu Pro Ala

130 135 140

Asn Ala Glu Ala Gln Thr Pro Ser Tyr Pro Asn Tyr Pro Pro Val Gln

145 150 155 160

Ala Pro Ile Phe Asn Pro Leu Asn Ser Ile Asn Asn Val Lys Thr Asn

165 170 175

Ser Leu Asn Val Gly Pro Ala Glu Phe Met Asn Thr Leu Asp Gly Ser

180 185 190

Ile Pro Ser Leu Ala Pro Ser Asn Ala Ile Gln Thr Asp Arg Ser Gly

195 200 205

Arg Pro Gln Pro Arg Tyr Lys Leu Gln Trp Arg Arg Phe Trp Trp Gly

210 215 220

Val Phe Ile Gln Gln Leu Ser Trp Met Ser Ala Ala Val Ser Thr

225 230 235