阅读说明：本技术 黔北麻羊timp1基因真核表达载体的构建方法 (Construction method of eukaryotic expression vector of TIMP1 gene of northern Guizhou goat ) 是由 陈祥 洪磊 唐文 周志楠 敖叶 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种黔北麻羊TIMP1基因真核表达载体的构建方法。本发明能在构建黔北麻羊TIMP1基因真核表达载体时,免除繁琐步骤,简化操作,节省科研成本。本发明通过简单的方法克隆黔北麻羊TIMP1基因并构建真核表达载体,为黔北麻羊基因检测提供基础方法,便于从基因水平研究其对黔北麻羊繁殖相关性状的影响,为黔北麻羊产羔性状的分子机制提供理论依据。(The invention discloses a construction method of a TIMP1 gene eukaryotic expression vector of a Qianbei goat. The invention can avoid fussy steps, simplify the operation and save the scientific research cost when constructing the eukaryotic expression vector of the TIMP1 gene of the Qianbei ma sheep. The invention clones the TIMP1 gene of the Qianbei ma sheep and constructs a eukaryotic expression vector by a simple method, provides a basic method for detecting the gene of the Qianbei ma sheep, is convenient to research the influence of the gene on the reproduction-related traits of the Qianbei ma sheep from the gene level, and provides a theoretical basis for the molecular mechanism of the lamb-bearing traits of the Qianbei ma sheep.)

1. A construction method of a TIMP1 gene eukaryotic expression vector of a northern Guizhou goat is characterized by comprising the following steps:

1) extracting RNA of the ovary tissue of the northern Guizhou goat, obtaining cDNA of the ovary tissue of the northern Guizhou goat through reverse transcription, obtaining a CDS region sequence of a TIMP1 gene through PCR amplification of the cDNA, recovering an amplified product gel, connecting the recovered product gel with a pMD19-T carrier, converting the product gel into TOP10 competent cells for culture, screening blue and white spots, selecting white colonies, performing amplification culture through an LB (LB) liquid culture medium, performing PCR detection and verification, and performing plasmid extraction sequencing verification; recovering the recombinant plasmid and the target fragment of the pEGFP-N3 vector through double enzyme digestion, and connecting to obtain a pEGFP-N3(+) -TIMP1 recombinant vector;

2) carrying out electrophoresis detection on the PCR product by using 1.5% agarose gel, cutting the gel, recovering a target band, and connecting the target band with a pMD19-T vector; a connection system: 1ul of pMD19-T vector, 5ul of target fragment, 1ul of buffer, 1ul of T4DNA ligase and 1ul of ddH2O 1; the reaction mixture was connected overnight in a metal bath at 16 ℃; connecting the reaction product to 100ul of competent cells, respectively adding white single colonies into an LB liquid culture medium containing AMP resistance after culture, performing shaking table shaking culture at 37 ℃ for 12-16 h to detect positive clones of a bacterial liquid by PCR, and extracting recombinant plasmids for sequencing verification;

3) the double enzyme digestion system is as follows: recombinant plasmid, pEGFP-N3(+) empty vector each 8ul, EcoRI 2ul, BamHI 2ul, 10 XQuickcut Green Buffer 2ul, ddH₂Supplementing O to 20ul, carrying out enzyme digestion in a constant-temperature water bath kettle at 37 ℃ for 3h, carrying out electrophoresis detection on the enzyme digestion product by using 1.5% agarose gel, cutting the agarose gel and recovering a target band and a pEGFP-N3(+) empty vector; a connection system: 10ul of pEGFP-N3(+) empty vector, 2ul of target fragment, 1.5ul of buffer and 1.5ul of T4DNA ligase; the reaction mixture was connected overnight in a metal bath at 16 ℃; linking the reaction product to 100uAnd l, culturing in competent cells, selecting white single colonies, respectively adding the white single colonies into LB liquid culture medium containing 5ul of sodium bicarbonate resistance, carrying out shaking culture on a shaking table at 37 ℃ for 12-16 h to detect positive clones, extracting recombinant plasmids, carrying out double enzyme digestion identification, and carrying out positive clones.

2. The construction method of the eukaryotic expression vector of TIMP1 gene in Qianbei ma sheep according to claim 1, which is characterized in that: the primer comprises an upstream primer EcoRI and a downstream primer BamHI, wherein the sequence of the upstream primer EcoRIF

3. The construction method of the eukaryotic expression vector of TIMP1 gene in Qianbei ma sheep according to claim 1, which is characterized in that: the PCR reaction system is as follows: the total reaction system of PCR amplification is 20ul, wherein each of the upstream primer and the downstream primer is 1.0 uL, the template cDNA1.0 uL, 2 XEs Taq MasterMix 10uL and ddH2O 7 uL, and the reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 5 min; { denaturation at 95 ℃ for 30s, annealing at 56.6 ℃ for 15s, and extension at 72 ℃ for 30s }. times.35 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C.

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of a eukaryotic expression vector based on TIMP1 gene of Qianbei ma sheep.

Background

Matrix metalloproteinase inhibitors (TIMPs) are an endogenous inhibitor of metalloproteinase activity, and were originally thought to act primarily to regulate matrix metalloproteinase activity and inhibit extracellular matrix transformation, including regulation of cell proliferation, differentiation, apoptosis, etc., and to play a critical role in the homeostasis of the extracellular matrix (Lambert et al, 2004). The TIMP1 gene was found in human skin fibroblasts, human serum, and bovine cartilage and aorta extracts cultured in vitro as collagenase inhibitors in the early 70's 20 th century. It has been found by researchers that the TIMP1 gene is involved in regulating the mouse reproductive cycle, and acts mainly on the uterus and ovary (Nothnick et al, 2000). More importantly, the TIMP1 gene is also considered to be a regulator of steroidogenesis (Boujard et al, 1995). On sheep, the expression of TIMP1 gene in the uterus was found to be influenced by hormones, such as estradiol, regulation (Hampton et al, 1995). On the other hand, in goats, Jiayin Peng et al (2015) research finds that the TIMP1 gene can increase the proliferation of goat oviduct epithelial cells, and shows that the TIMP1 gene plays an important role in promoting the survival of the oviduct epithelial cells. Therefore, the function of TIMP1 gene is not limited to be used as an inhibitor of matrix metalloproteinase, and the important function of TIMP1 gene on animal reproduction is gradually noticed, so that a new idea can be provided for the research of improving animal reproduction performance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method of a eukaryotic expression vector of TIMP1 gene of Qianbei ma sheep.

The invention is realized by the following steps: the construction method of the eukaryotic expression vector of the TIMP1 gene of the northern Guizhou goat comprises the following steps:

1) extracting RNA of the ovary tissue of the northern Guizhou goat, obtaining cDNA of the ovary tissue of the northern Guizhou goat through reverse transcription, obtaining a CDS region sequence of a TIMP1 gene through PCR amplification of the cDNA, recovering an amplified product gel, connecting the recovered product gel with a pMD19-T carrier, converting the product gel into TOP10 competent cells for culture, screening blue and white spots, selecting white colonies, performing amplification culture through an LB (LB) liquid culture medium, performing PCR detection and verification, and performing plasmid extraction sequencing verification; recovering the recombinant plasmid and the target fragment of the pEGFP-N3 vector through double enzyme digestion, and connecting to obtain a pEGFP-N3(+) -TIMP1 recombinant vector;

2) carrying out electrophoresis detection on the PCR product by using 1.5% agarose gel, cutting the gel, recovering a target band, and connecting the target band with a pMD19-T vector; a connection system: 1ul of pMD19-T vector, 5ul of target fragment, 1ul of buffer, 1ul of T4DNA ligase and 1ul of ddH2O 1; the reaction mixture was connected overnight in a metal bath at 16 ℃; connecting the reaction product to 100ul of competent cells, respectively adding white single colonies into an LB liquid culture medium containing AMP resistance after culture, performing shaking culture on a table at 37 ℃ for 12 h-16 hPCR detection bacteria liquid positive clones, and extracting recombinant plasmid sequencing for verification;

3) the double enzyme digestion system is as follows: recombinant plasmid, pEGFP-N3(+) empty vector each 8ul, EcoRI 2ul, BamHI 2ul, 10 XQuickcut Green Buffer 2ul, ddH₂Supplementing O to 20ul, carrying out enzyme digestion in a constant-temperature water bath kettle at 37 ℃ for 3h, carrying out electrophoresis detection on the enzyme digestion product by using 1.5% agarose gel, cutting the agarose gel and recovering a target band and a pEGFP-N3(+) empty vector; a connection system: 10ul of pEGFP-N3(+) empty vector, 2ul of target fragment, 1.5ul of buffer and 1.5ul of T4DNA ligase; the reaction mixture was connected overnight in a metal bath at 16 ℃; connecting the reaction product to 100ul of competent cells, selecting white single colonies after culture, respectively adding the white single colonies into LB liquid culture medium containing 5ul of sodium bicarbonate resistance, carrying out shaking culture on a shaker at 37 ℃ for 12-16 h to detect positive clones, extracting recombinant plasmids, carrying out double enzyme digestion identification, and carrying out positive clones.

The primer comprises an upstream primer EcoRI and a downstream primer BamHI, wherein the sequence 5' of the upstream primer EcoRIFCCG ATGGCCCTCTTTGCACCCAT-3'; the single underlined part is a protection base, and the double underlined part is a restriction enzyme site; sequence of the downstream primer BamHI R: 5' -GC

TCAGGCCCCCCGGGGCCGCA-3'; the single underlined part is the protecting base and the double underlined part is the cleavage site.

The PCR reaction system is as follows: the total reaction system for PCR amplification is 20ul, wherein each of the upstream primer and the downstream primer is 1.0 mu L, the template cDNA is 1.0 mu L, 2 XEs Taq MasterMix is 10 mu L, ddH2O 7 mu L, and the reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 5 min; { denaturation at 95 ℃ for 30s, annealing at 56.6 ℃ for 15s, and extension at 72 ℃ for 30s }. times.35 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C.

Due to the adoption of the technical scheme, compared with the prior art, the construction method disclosed by the invention can be used for avoiding complicated steps, simplifying the operation and saving the scientific research cost when constructing the eukaryotic expression vector of the TIMP1 gene of the Qianbei ma goat. The invention clones the TIMP1 gene of the Qianbei ma sheep and constructs a eukaryotic expression vector by a simple method, provides a basic method for detecting the gene of the Qianbei ma sheep, is convenient to research the influence of the gene on the reproduction-related traits of the Qianbei ma sheep from the gene level, and provides a theoretical basis for the molecular mechanism of the lamb-bearing traits of the Qianbei ma sheep.

Drawings

FIG. 1 is an amplification product of CDS region of TIMP1 gene;

FIG. 2 shows PCR results of bacterial suspension of TIMP1 gene;

FIG. 3 shows the results of gel electrophoresis of the TIMP1PFGFP-N3(+) recombinant plasmid;

FIG. 4 shows the double digestion of the recombinant plasmid PFGFP-N3(+) -TIMP 1.

Detailed Description

Example 1 of the invention: the animal of the embodiment is from Fuxing animal husbandry Limited company in the prefecture of Water-learning county of Guizhou province, three healthy (2-3 years old) female sheep of the Qianbei ma sheep are selected to be slaughtered, ovary tissues are collected and stored in liquid nitrogen for extracting total RNA of the tissues.

The main reagents required in this example are: reverse transcription kit, agarose, high-purity plasmid miniextraction kit, gel recovery kit and the like are purchased from Beijing kang, a century Biotechnology Co., Ltd; t-clone PCR product cloning kits and the like are purchased from Shanghai biological engineering technology, Inc., Goldview dye 2 XTaq Master Mix, agarose, TOP10 competent cells, LB culture medium, ampicillin and the like are purchased from Beijing Ding national biological engineering technology, Inc., and the like. DM2000DNA Marker, restriction enzymes EcoRI, BamHI and T4DNA ligase were purchased from Dalibao bioengineering, Inc.

Extraction of RNA (ribonucleic acid) of ovary tissue of northern Guizhou goat

(1) Adding liquid nitrogen into about 100mg of tissue sample in a mortar, grinding into powder, transferring into a 1.5ml centrifuge tube containing 1ml Trizol, turning upside down, mixing, standing for 10min, adding 200 μ l chloroform, and standing on ice for 3 min;

(2) centrifuging at 12000rpm at 4 deg.C for 15min, transferring supernatant to a new centrifuge tube with a pipette gun, adding 500 μ l isopropanol, mixing, standing on ice for 10min, centrifuging at 12000rpm at 4 deg.C for 10 min;

(3) the supernatant was discarded, the white precipitate (RNA as a white precipitate) was retained, 1ml of 75% ethanol was added, and the mixture was allowed to stand on ice for 2min, at 4 ℃ and 7500rpm, and centrifuged for 10 min. Repeating the step once;

(4) the supernatant was discarded, and the tube was placed upside down on filter paper and allowed to air dry for 10 min. Adding 30 μ l DEPC water, and incubating in incubator at 55-60 deg.C for 15 min;

(5) and (3) taking 1 mu l of RNA to carry out concentration and purity detection in an ultramicro ultraviolet spectrophotometer, wherein the OD260/OD280 value is 1.8-2.0, the effect is optimal, and storing the RNA in a refrigerator at the temperature of-80 ℃ for later use.

Reverse transcription reaction of RNA

And synthesizing the first strand cDNA of the extracted ovarian tissue RNA with the quality meeting the standard by using a reverse transcription kit. Carrying out reverse transcription on the extracted RNA into cDNA according to a 20-microliter system, wherein the system comprises the following steps: adding dNTP Mix 4 uL, RT Buffer 4 u L, primerMix 2u L, RNA template 2u L, DTT 2u L, HiFiscript 1uL and RNase-Free Water 5uL into a PCR tube, oscillating and mixing uniformly, centrifuging, and incubating for 50min at 42 ℃ and 5min at 85 ℃ on a PCR instrument; after the reaction is finished, 1 mu L of reaction product is taken to be put into an ultramicro ultraviolet spectrophotometer to carry out concentration and purity detection, and is stored at-20 ℃ for standby.

Primer design of TMP1 Gene

Primers for TIMP1 gene (Primier5.0 software) were designed based on CDS region sequence of LYRM1 gene uploaded from Genebank, and synthesized by Biotechnology engineering (Shanghai) GmbH, and primer sequence information of TIMP1 gene is shown in Table 1.

TABLE 1 primer sequence information of TIMP1 Gene

Amplification of CDS region of TIMP1 Gene

Taking cDNA obtained by reverse transcription as a template to carry out TIMP1 gene CDS region amplification, wherein the total reaction system is 20ul, the upstream primer and the downstream primer are respectively 1.0 mu L (10 mu M), the template cDNA is 1.0 mu L, 2 XEsTaq MasterMix is 10 mu L, ddH2O 7 mu L, and the reaction conditions of PCR are as follows: pre-denaturation at 95 ℃ for 5 min; { denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 15s, and extension at 72 ℃ for 30s }. times.35 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C. The PCR amplification products were detected by electrophoresis on a 1.5% agarose gel.

Recovery and purification of amplification product of CDS region of TIMP1 gene

(1) Cutting the agarose gel containing the target fragment, putting the agarose gel into a weighed 1.5ml centrifuge tube, and weighing the gel;

(2) adding 3 volumes of Buffer DE-A, mixing uniformly, placing in a 75 ℃ water bath kettle, heating to completely melt the gel;

(3) adding 0.5 volume of Buffer DE-A volume of Buffer DE-B, and mixing uniformly;

(4) transferring the mixed solution in the step 3 into a DNA preparation tube, centrifuging at 12000rpm for 1min, and pouring off the filtrate; adding 500 mu l of buffer W1, centrifuging for 30s at 12000g, and removing the filtrate;

(5) adding 700 μ l buffer W2, centrifuging at 12000rpm for 1min, discarding the filtrate, and repeating the step once;

(6) placing the prepared tube back into a 2ml centrifuge tube, and centrifuging for 2min at 12000g air;

(7) placing the prepared tube into a 1.5ml centrifuge tube, adding 30 ul Eluent, standing at room temperature for 5min, and centrifuging at 12000rpm for 1min to elute DNA;

(8) using an Eluent correction ultramicro ultraviolet spectrophotometer to detect the concentration and purity of the gel recovered product, and storing the gel recovered product in a refrigerator at the temperature of-20 ℃;

the CDS region amplification gel recovery product is connected with a Pucm-T carrier

1ul of PUCm-T vector (50ng) was sequentially added to a PCR tube, 5. mu.L of a target band-recovered product, 10 XLigationBuffer 1. mu.L, 50% PEG 40001. mu.L, Sterilized ddH2O 1. mu.L, and T4DNA Ligase 1. mu.L were mixed well to construct a ligation reaction system of 10. mu.L, and the ligation was performed overnight in a metal bath at 16 ℃.

Plasmid transformation and blue-white screening

And (3) plasmid transformation:

(1) thawing 100ul of competent cells on ice, and gently suspending the cells after complete thawing;

(2) adding the 5ul connecting solution, mixing, and standing on ice for 30 min;

(3) heating in 42 deg.C water bath for 90s, and standing on ice for 20 min;

(4) adding 700ul LB culture medium, shaking and culturing for 1h at 37 ℃ by a shaking table at 200 rpm;

(5) centrifuging at 4000rpm for 5min, sucking off 500ul of supernatant with a pipette, and suspending the cells with the rest of the culture medium;

(6) the bacterial suspension was spread evenly on LB plates containing 100ul ampicillin resistance;

(7) the plates were first placed in the forward direction for 1 hour to absorb excess fluid and then incubated overnight in an inverted position.

Screening

Observing the growth condition of colonies on an ampicillin-resistant LB plate in a clean bench, picking single white single colonies beside flame by using a pipette, respectively adding the single white single colonies into an LB liquid culture medium containing AMP resistance, and carrying out shake culture on a shaking table at 37 ℃ for 12-16 h.

PCR identification of bacterial liquid

And (3) carrying out PCR amplification by using the cultured bacterial liquid as a template according to the following system and conditions, and preliminarily detecting the construction condition of the carrier. And (3) PCR reaction system: the upstream and downstream primers were 1.0. mu.L each, template 1.0. mu.L, 2 XEs Taq MasterMix 10. mu.L, ddH2O supplemented to 20. mu.L. The reaction conditions of PCR were: pre-denaturation at 95 ℃ for 5 min; { denaturation at 95 ℃ for 30s, annealing at 57 ℃ for 15s, and extension at 72 ℃ for 30s }. times.35 cycles; final extension at 72 deg.C for 5min, and storage at 4 deg.C. The PCR amplification product was detected by electrophoresis on a 1.5% agarose gel and the band of interest was visualized by a gel imaging system.

Extraction of recombinant plasmid

Extracting plasmids of a bacterial liquid with correct PCR identification according to the specification of a Kangji century high-purity plasmid miniextraction kit, and operating according to the following steps:

(1) 2ml of overnight-cultured bacterial liquid is added into a centrifuge tube, centrifuged at 6200 Xg for 3min to collect bacterial precipitates, and the supernatant is removed as much as possible.

(2) 250ul of Buffer P1 (added with RNase A) was added to the pellet, and the pellet was mixed thoroughly with a vortex shaker to suspend the pellet and ensure thorough mixing of the pellet.

(3) 250ul of Buffer P2 was added to the tube, and the mixture was gently inverted from top to bottom and mixed 4-6 times, and mixed well to lyse the cells, at which time the solution was cool and viscous. The mixture is gently mixed without violent shaking so as to avoid the pollution of the genome DNA, and the time used in the step is not more than 5min so as to avoid the plasmid damage.

(4) 350ul of buffer N3 was added to the tube and mixed by immediately and gently turning upside down for 4-6 times, and mixed well until white flocculent precipitate appeared, and centrifuged at 12000rpm for 10 min. Note that Buffer N3 was added immediately prior to mixing to avoid local precipitation.

(5) Transferring the supernatant obtained in the step (4) into an adsorption column filled into a collecting pipe, centrifuging at 12000rpm for 1min, pouring waste liquid in the collecting pipe, and replacing the adsorption column into the collecting pipe again.

(6) 750ul of Buffer PW (to which absolute ethanol has been added) was added to the adsorption column, centrifuged at 12000rpm for 1min, and the waste liquid in the collection tube was decanted.

(7) The column was replaced into the collection tube, centrifuged at 12000rpm for 2min, the waste liquid was decanted, the column was left at room temperature for several minutes to remove the remaining ethanol, and the collected plasmid was stored at-20 ℃ for further use. The successfully cloned plasmids are selected and sent to the company Limited of the engineering bioengineering (Shanghai) for sequencing.

Double digestion of recombinant plasmid and pEGFP-N3(+) empty vector

Carrying out EcoRI and BamHI double enzyme digestion on the recombinant plasmid and the PEGFP-N3(+) empty vector, wherein the enzyme digestion system is as follows;

carrying out enzyme digestion for 4h in a constant-temperature water bath at 37 ℃, carrying out electrophoresis detection on the enzyme digestion product by using 1% agarose gel, carrying out gel recovery on a target band and a large fragment of a pEGFP-N3(+) empty vector according to the specification of a SanPrep column type DNA gel recovery kit, and carrying out a connection reaction according to the following system and conditions:

cloning and purification of pEGFP-N3(+) -TIMP1 recombinant vector

Cloning and purifying the pEGFP-N3(+) -TIMP1 recombinant vector, respectively carrying out bacterial liquid PCR identification and plasmid extraction, carrying out double enzyme digestion identification on the recombinant plasmid, and sending the bacterial liquid with correct identification to Shanghai biological Limited company for bidirectional sequencing.

Preservation of recombinant vector in bacterial liquid

Adding the correctly identified bacterial liquid of the recombinant plasmid into a 2ml centrifuge tube, adding 50% of glycerol in equal proportion, turning upside down and mixing uniformly, sealing with a PV membrane, and storing in a refrigerator at-80 ℃.

Sequence listing

<110> Guizhou university

Construction method of <120> TIMP1 gene eukaryotic expression vector of Qianbei ma sheep

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>29

<212>DNA

<213> goat (Capra hircus)

<400>1

ccggaattca tggccctctt tgcacccat 29

<210>2

<211>28

<212>DNA

<213> goat (Capra hircus)

<400>2

gcggatcctc aggccccccg gggccgca 28