阅读说明：本技术 一种鸡白痢沙门菌噬菌体裂解酶的发酵制备方法 (Fermentation preparation method of chicken pullorum salmonella bacteriophage lyase ) 是由 冯新 宋战昀 于 2021-07-26 设计创作，主要内容包括：本发明属于生物学技术领域,公开了一种鸡白痢沙门菌噬菌体裂解酶的发酵制备方法,包括：进行鸡白痢沙门菌、沙门菌噬菌体YSP2的培养,并提取沙门菌噬菌体YSP2基因组；以噬菌体YSP2的全基因组为模板,利用引物LySP2-ZF/LySP2-ZR对裂解酶目的基因LySP2进行PCR扩增；进行表达载体pPICZ-LySP2的构建与鉴定,进行重组毕赤酵母菌株X33-pPICZ-LySP2的筛选和鉴定；同时进行重组毕赤酵母菌X33-pPICZ-LySP2的诱导发酵及条件优化。本发明构建了重组裂解酶LySP2基因的毕赤酵母表达菌株X33-pPICZ-LySP2。(The invention belongs to the technical field of biology, and discloses a fermentation preparation method of a chicken pullorum salmonella bacteriophage lyase, which comprises the following steps: culturing Salmonella pullorum and Salmonella phage YSP2, and extracting the genome of Salmonella phage YSP 2; carrying out PCR amplification on a target gene LySP2 of the lyase by using a primer LySP2-ZF/LySP2-ZR and taking the whole genome of a bacteriophage YSP2 as a template; constructing and identifying an expression vector pPICZ-LySP2, and screening and identifying a recombinant Pichia pastoris strain X33-pPICZ-LySP 2; and simultaneously carrying out induced fermentation and condition optimization on the recombinant pichia pastoris X33-pPICZ-LySP 2. The invention constructs a Pichia pastoris expression strain X33-pPICZ-LySP2 of a recombinant lyase LySP2 gene.)

1. A fermentation preparation method of Salmonella pullorum phage lyase is characterized by comprising the following steps:

culturing Salmonella pullorum and Salmonella phage YSP2, and extracting the genome of Salmonella phage YSP 2;

carrying out PCR amplification on a target gene LySP2 of the lyase by using a primer LySP2-ZF/LySP2-ZR and taking the whole genome of a bacteriophage YSP2 as a template;

constructing and identifying an expression vector p PICZ-LySP2, and screening and identifying a recombinant Pichia pastoris strain X33-p PICZ-LySP 2; and simultaneously carrying out induced fermentation and condition optimization on the recombinant pichia pastoris X33-p PICZ-LySP 2.

2. The method for preparing Salmonella pullorum phage lyase by fermentation according to claim 1, wherein the culturing Salmonella pullorum phage YSP2 comprises:

the culture of the salmonella pullorum comprises the following steps:

inoculating loop, selecting Salmonella pullorum on LB plate, streaking, and culturing at 37 deg.C overnight; selecting a single colony to be inoculated into an LB liquid culture medium, and carrying out shake culture at 37 ℃ and 160r/min until the concentration of the colony is 108 CFU/mL;

the performing of the culture of the salmonella phage YSP2 comprises:

selecting host bacteria, inoculating the host bacteria into 5mL LB liquid culture medium, and performing shake culture at 37 ℃ and 160r/min until OD600 is 0.6; adding 100 mu L of phage liquid, shaking and culturing at 37 ℃ and 160r/min until the culture solution is clear and transparent; filtering the culture solution with 0.22 μm microporous membrane, collecting filtrate, adding 30% glycerol, and subpackaging into 1.5mL centrifuge tubes, and storing at-4 deg.C and-80 deg.C respectively.

3. The method for preparing Salmonella pullorum phage lyase by fermentation according to claim 1, wherein the extracting Salmonella phage YSP2 genome comprises: respectively adding 20 mu L of LProteinase K and 200 mu L of sample into a 1.5m L centrifuge tube and mixing uniformly; adding 200 mu LBB, vortexing and shaking for 15s, and incubating for 15min at 56 ℃; adding 250 μ L of anhydrous ethanol, vortex and shake for 15s, standing at room temperature for 5 min; centrifuging at 10000r/min for 1min, and discarding waste liquid; adding 500 mu LWB, centrifuging at 10000r/min for 1min, discarding the waste liquid, and repeating once; centrifuging at 10000r/min for 1min for air drying, and transferring to a new 1.5mL RNase-free centrifuge tube; adding 20 μ L sterile water, centrifuging at room temperature 10000r/min for 1min, eluting, and collecting DNA, and storing at-20 deg.C for use.

4. The method for preparing Salmonella pullorum phage lyase by fermentation according to claim 1, wherein the primer LySP2-ZF/LySP2-ZR is designed according to the sequence of Salmonella pullorum phage YSP2 lyase Ly SP2 gene, and is obtained by inserting restriction sites of restriction enzymes Xho I and Xba I into 5 'end and 3' end, and is used for cloning lyase LySP2 gene and constructing eukaryotic expression vector;

the theoretical amplification fragment size of the primer LySP2-ZF/LySP2-ZR is 495 bp; the sequence of the primer LySP2-ZF is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the primer LySP2-ZR is shown as SEQ ID NO: 2, respectively.

5. The method for preparing Salmonella pullorum phage lyase by fermentation according to claim 1, wherein the construction and identification of the expression vector p PICZ-LySP2 comprises:

(1) the double enzyme digestion yeast expression vector p PICZ-alpha A is connected with the LySP2 gene fragment by utilizing T4 DNA ligase; transforming the ligation product into a competent DH5 alpha, screening a positive transformant, extracting a plasmid, and performing PCR and sequencing identification;

(2) selecting the transformed single bacterium to fall into 5mL LB culture medium containing Zeocin + low salt, culturing for 6-8 hours at 37 ℃, carrying out PCR identification by taking bacterium liquid as a template, and carrying out amplification by utilizing a PCR Mix system, wherein the primers used for the identification are a universal primer 5' AOX on a p PICZ-alpha A carrier and a target gene downstream primer LySP 2-ZR;

(3) extracting plasmids, carrying out double enzyme digestion identification, carrying out 1% gel electrophoresis identification, carrying out positive plasmids, carrying out sequencing, carrying out comparison analysis on a sequencing result, and detecting whether base mutation and frame shift exist.

6. The method for preparing bacteriophage lytic enzyme of salmonella pullorum according to claim 5, wherein in the step (2), the PCR amplification process comprises: pre-denaturation at 94 ℃ for 10min for 1 cycle; pre-denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 35s, extension at 72 ℃ for 40s, and reaction for 29 cycles; extension at 72 ℃ for 10min and at 4 ℃ for 10 min.

7. The method for preparing phage lyase for pullorum disease of chicken as claimed in claim 1, wherein the screening and identification of recombinant pichia pastoris strain X33-p PICZ-LySP2 comprises: selecting a single colony to YPD culture medium containing Zeocin +, and culturing at 29 ℃ for 12 h; adding 1mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 8000r/min for 5min, and collecting thalli; 50 μ L of ddH pH 8.0₂Dissolving DNA with O, and storing at-20 ℃ for later use; the correct positive recombinant yeast strains identified by PCR were designated X33-pPICZ-LySP2 and the empty yeast strain X33-p PICZ.

8. The method for preparing bacteriophage lytic enzyme of salmonella pullorum according to claim 1, wherein the inducing fermentation and condition optimization of the recombinant pichia pastoris X33-p PICZ-LySP2 comprise:

1) selecting transformants which are positive by yeast colony PCR on the transformed YPD plates containing Zeocin + and inoculating the transformants into 25mLBMGY liquid culture medium;

2) culturing at 29 deg.C and 350r/min with shaking until OD600 is 2.0; collecting part of the culture solution, centrifuging at room temperature of 6000r/min for 5min, removing supernatant, and washing thallus with sterile PBS for 2 times;

3) transferring the thallus to a 50mLBMMY culture medium, controlling the initial OD600 to be 1.0, carrying out shake culture at 29 ℃ and 350r/min for 72-96h, supplementing 1% methanol to the culture medium every 12h, sampling every 24h, analyzing the thallus growth condition, and screening high-expression strains.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fermentation preparation method of a chicken pullorum salmonella bacteriophage lyase.

Background

At present, pullorum disease is one of 14 animal epidemic diseases which are required to be monitored in the Ministry of agriculture in China, and the damage of pathogenic bacteria to poultry severely restricts the development of poultry industry. The purification of pullorum disease in chicken farms is the best method for eradicating the disease. Antibiotics are traditional medicines for preventing and treating pullorum disease, but because of long-term wide use and even abuse of antibiotics, drug resistance is increasingly serious, so that prevention and treatment and elimination of pullorum disease are increasingly difficult. The bacteriophage and the lyase thereof are widely researched and applied to the aspects of improving food, medical safety and the like as a novel antimicrobial preparation, so that the screening and identification of the bacteriophage and the lyase thereof of the high-efficiency salmonella pullorum have important significance for preventing and purifying pullorum.

Through the above analysis, the problems and defects of the prior art are as follows: the currently prepared salmonella pullorum bacteriophage lyase has poor cracking effect, poor bacteriostatic effect and poor stability.

The difficulty in solving the above problems and defects is: the salmonella pullorum bacteriophage is inconvenient to use clinically, especially difficult to prepare in large scale, and the use of the salmonella pullorum bacteriophage lyase can not only keep the activity, but also has the potential of large-scale fermentation production.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a fermentation preparation method of a chicken pullorum salmonella bacteriophage lyase.

The invention is realized in such a way that the fermentation preparation method of the salmonella pullorum bacteriophage lyase comprises the following steps:

culturing salmonella pullorum and a salmonella phage YSP2, and extracting a salmonella phage YSP2 genome;

secondly, carrying out PCR amplification on a target gene LySP2 of the lyase by using a primer LySP2-ZF/LySP2-ZR and taking the whole genome of the phage YSP2 as a template;

step three, constructing and identifying an expression vector p PICZ-LySP2, and screening and identifying a recombinant Pichia pastoris strain X33-p PICZ-LySP 2; and simultaneously carrying out induced fermentation and condition optimization on the recombinant pichia pastoris X33-pPICZ-LySP 2.

Further, in the first step, the culturing salmonella pullorum and the salmonella phage YSP2 comprises:

the culture of the salmonella pullorum comprises the following steps:

inoculating loop, selecting Salmonella pullorum on LB plate, streaking, and culturing at 37 deg.C overnight; selecting a single colony to be inoculated into an LB liquid culture medium, and carrying out shake culture at 37 ℃ and 160r/min until the concentration of the colony is 108 CFU/mL;

the performing of the culture of the salmonella phage YSP2 comprises:

selecting host bacteria, inoculating the host bacteria into 5mL LB liquid culture medium, and performing shake culture at 37 ℃ and 160r/min until OD600 is 0.6; adding 100 mu L of phage liquid, shaking and culturing at 37 ℃ and 160r/min until the culture solution is clear and transparent; filtering the culture solution with 0.22 μm microporous membrane, collecting filtrate, adding 30% glycerol, and subpackaging into 1.5mL centrifuge tubes, and storing at-4 deg.C and-80 deg.C respectively.

Further, in the first step, the extracting of the genome of the salmonella phage YSP2 comprises:

respectively adding 20 mu L of protease K and 200 mu L of sample into a 1.5mL centrifuge tube, and uniformly mixing; adding 200 mu LBB, vortexing and shaking for 15s, and incubating for 15min at 56 ℃; adding 250 μ L of anhydrous ethanol, vortex and shake for 15s, standing at room temperature for 5 min; centrifuging at 10000r/min for 1min, and discarding waste liquid; adding 500 microliter WB, centrifuging at 10000r/min for 1min, discarding the waste liquid, and repeating once; centrifuging at 10000r/min for 1min for air drying, and transferring to a new 1.5mL RNase-free centrifuge tube; adding 20 μ L sterile water, centrifuging at room temperature 10000r/min for 1min, eluting, and collecting DNA, and storing at-20 deg.C for use.

Further, in the second step, the primer LySP2-ZF/LySP2-ZR is designed according to the sequence of the Salmonella pullorum phage YSP2 lyase Ly SP2 gene, is obtained by inserting restriction enzyme sites of restriction enzymes Xho I and Xba I into the 5 'end and the 3' end, and is used for cloning the lyase LySP2 gene and constructing a eukaryotic expression vector;

the theoretical amplification fragment size of the primer LySP2-ZF/LySP2-ZR is 495 bp; the sequence of the primer LySP2-ZF is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the primer LySP2-ZR is shown as SEQ ID NO: 2, respectively.

Further, in step three, the construction and identification of the expression vector p PICZ-LySP2 comprise the following steps:

(1) the double enzyme digestion yeast expression vector p PICZ-alpha A is connected with the LySP2 gene fragment by utilizing T4 DNA ligase; the ligation product was transformed into competent DH5 α, positive transformants were selected and plasmids were extracted for PCR and sequencing identification.

(2) Selecting the transformed single bacterium to fall into 5mL LB culture medium containing Zeocin + low salt, culturing for 6-8 hours at 37 ℃, carrying out PCR identification by taking bacterium liquid as a template, and carrying out amplification by utilizing a PCR Mix system, wherein the primers used for the identification are a universal primer 5' AOX on a p PICZ-alpha A carrier and a target gene downstream primer LySP 2-ZR;

(3) extracting plasmids, carrying out double enzyme digestion identification, carrying out 1% gel electrophoresis identification, carrying out positive plasmids, carrying out sequencing, carrying out comparison analysis on a sequencing result, and detecting whether base mutation and frame shift exist.

Further, in the step (2), the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 10min for 1 cycle; pre-denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 35s, extension at 72 ℃ for 40s, and reaction for 29 cycles; extension at 72 ℃ for 10min and at 4 ℃ for 10 min.

Further, in step three, the screening and identification of the recombinant pichia pastoris strain X33-p PICZ-LySP2 comprises:

selecting a single colony to YPD culture medium containing Zeocin +, and culturing at 29 ℃ for 12 h; adding 1mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 8000r/min for 5min, and collecting thalli; 50 μ L of ddH pH 8.0₂Dissolving DNA with O, and storing at-20 ℃ for later use; the correct positive recombinant yeast strains identified by PCR were designated X33-p PICZ-LySP2 and the empty yeast strain X33-p PICZ.

Further, in the third step, the induction fermentation and condition optimization of the recombinant pichia pastoris X33-p PICZ-LySP2 comprise the following steps:

1) transformants identified as positive by yeast colony PCR on transformed YPD plates containing Zeocin + were picked and inoculated into 25mL BMGY liquid medium;

2) culturing at 29 deg.C and 350r/min with shaking until OD600 is 2.0; collecting part of the culture solution, centrifuging at room temperature of 6000r/min for 5min, removing supernatant, and washing thallus with sterile PBS for 2 times;

3) transferring the thallus to a 50mLBMMY culture medium, controlling the initial OD600 to be 1.0, carrying out shake culture at 29 ℃ and 350r/min for 72-96h, supplementing 1% methanol to the culture medium every 12h, sampling every 24h, analyzing the thallus growth condition, and screening high-expression strains.

By combining all the technical schemes, the invention has the advantages and positive effects that: the invention constructs a Pichia pastoris expression strain X33-p PICZ-LySP2 of a recombinant lyase LySP2 gene, and the methanol is used for inducing the recombinant lyase LySP2 with secretory expression and antibacterial activity, wherein the diameter of an antibacterial ring of the recombinant lyase LySP2 can reach 30.15mm, and the expression amount can reach 238.76 mu g/mL.

The recombinant lyase LySP2 has good thermal stability and wide tolerance range to acid and alkali. The enzyme activity is kept above 85 percent when the enzyme acts for 30min under the condition of pH value of 3-9, the enzyme activity is basically not lost when the enzyme acts for 30min under the temperature of 4-40 ℃, and the enzyme activity has better application prospect.

The recombinant lyase LySP2 has a lysis effect on salmonella and also has a lysis activity on Escherichia coli, and is wider than the lysis spectrum of phage YSP 2. In vivo experiments prove that the protection rate of the recombinant lyase LySP2 on a chicken pullorum salmonella infection model is 70%, the clearance rate of the liver salmonella can reach 100%, and the clearance rate of the intestinal salmonella can reach 50%.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flow chart of a fermentation preparation method of Salmonella pullorum bacteriophage lyase provided in the embodiment of the present invention.

FIG. 2 is a schematic diagram of a phage whole genome provided in an embodiment of the invention.

FIG. 3 is a schematic diagram of the PCR and enzyme digestion identification results of the recombinant plasmid p PICZ-LySP2 provided by the embodiment of the invention.

FIG. 4 is a diagram illustrating the results of activity assays of fermentation products provided by embodiments of the present invention.

Fig. 5 is a schematic diagram of the therapeutic effect of LySP2 on pullorum disease infection model provided by the embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a fermentation preparation method of a chicken pullorum salmonella bacteriophage lyase, and the invention is described in detail with reference to the attached drawings.

The yeast X33-p PICZ-LySP2 is a academic paper, and the expression of Salmonella pullorum phage lyase LySP2 in Pichia pastoris and the primary application thereof are disclosed in Zhang poplar, 2018, Jilin university.

As shown in FIG. 1, the fermentation preparation method of Salmonella pullorum bacteriophage lyase provided by the embodiment of the invention comprises the following steps:

s101, culturing salmonella pullorum and salmonella phage YSP2, and extracting a salmonella phage YSP2 genome;

s102, carrying out PCR amplification on a target gene LySP2 of the lyase by using a primer LySP2-ZF/LySP2-ZR and taking the whole genome of a phage YSP2 as a template;

s103, constructing and identifying an expression vector p PICZ-LySP2, and screening and identifying a recombinant Pichia pastoris strain X33-p PICZ-LySP 2; and simultaneously carrying out induced fermentation and condition optimization on the recombinant pichia pastoris X33-p PICZ-LySP 2.

The fermentation preparation method of Salmonella pullorum phage lyase provided by the invention can be implemented by other steps by persons of ordinary skill in the art, and the fermentation preparation method of Salmonella pullorum phage lyase provided by the invention shown in FIG. 1 is only one specific example.

In step S101, the culturing salmonella pullorum and the salmonella phage YSP2 provided in the embodiment of the present invention includes:

the culture of salmonella pullorum comprises the following steps:

inoculating loop, selecting Salmonella pullorum on LB plate, streaking, and culturing at 37 deg.C overnight; selecting a single colony to be inoculated into an LB liquid culture medium, and carrying out shake culture at 37 ℃ and 160r/min until the concentration of the colony is 108 CFU/mL;

performing the culturing of the salmonella phage YSP2 comprises:

selecting host bacteria, inoculating the host bacteria into 5mL LB liquid culture medium, and performing shake culture at 37 ℃ and 160r/min until OD600 is 0.6; adding 100 mu L of phage liquid, shaking and culturing at 37 ℃ and 160r/min until the culture solution is clear and transparent; filtering the culture solution with 0.22 μm microporous membrane, collecting filtrate, adding 30% glycerol, and subpackaging into 1.5mL centrifuge tubes, and storing at-4 deg.C and-80 deg.C respectively.

In step S101, the extracting of the genome of the salmonella phage YSP2 provided in the embodiment of the present invention includes:

respectively adding 20 mu L of protease K and 200 mu L of sample into a 1.5mL centrifuge tube, and uniformly mixing; adding 200 mu L of BB, performing vortex oscillation for 15s, and incubating for 15min at 56 ℃; adding 250 μ L of anhydrous ethanol, vortex and shake for 15s, standing at room temperature for 5 min; centrifuging at 10000r/min for 1min, and discarding waste liquid; adding 500 microliter WB, centrifuging at 10000r/min for 1min, discarding the waste liquid, and repeating once; centrifuging at 10000r/min for 1min for air drying, and transferring to a new 1.5mL RNase-free centrifuge tube; adding 20 μ L sterile water, centrifuging at room temperature 10000r/min for 1min, eluting, and collecting DNA, and storing at-20 deg.C for use.

In step S102, the primer LySP2-ZF/LySP2-ZR provided in the embodiment of the present invention is obtained by inserting restriction sites of restriction enzymes Xho I and Xba I into the 5 'end and the 3' end according to the sequence design of the LySP2 lyase Ly SP2 gene of salmonella pullorum, and is used for cloning the LySP2 gene and constructing a eukaryotic expression vector;

the theoretical amplified fragment size of the primer LySP2-ZF/LySP2-ZR is 495 bp;

the sequence of the primer LySP2-ZF is SEQ ID NO: 1: ccgc | tcgagaaaagaatggctattaaaaagacaatagccg-3';

the sequence of the primer LySP2-ZR is SEQ ID NO: 2: tgct | ctagatcatttattcagatccattacacaa-3'.

In step S103, the construction and identification of the expression vector p PICZ-LySP2 provided in the embodiment of the present invention includes:

(1) the double enzyme digestion yeast expression vector p PICZ-alpha A is connected with the LySP2 gene fragment by utilizing T4 DNA ligase; the ligation product was transformed into competent DH5 α, positive transformants were selected and plasmids were extracted for PCR and sequencing identification.

(2) Selecting the transformed single bacterium to fall into 5mL LB culture medium containing Zeocin + low salt, culturing for 6-8 hours at 37 ℃, carrying out PCR identification by taking bacterium liquid as a template, and carrying out amplification by utilizing a PCR Mix system, wherein the primers used for the identification are a universal primer 5' AOX on a p PICZ-alpha A carrier and a target gene downstream primer LySP 2-ZR;

(3) extracting plasmids, carrying out double enzyme digestion identification, carrying out 1% gel electrophoresis identification, carrying out positive plasmids, carrying out sequencing, carrying out comparison analysis on a sequencing result, and detecting whether base mutation and frame shift exist.

In step (2), the PCR amplification procedure provided in the embodiment of the present invention is as follows: pre-denaturation at 94 ℃ for 10min for 1 cycle; pre-denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 35s, extension at 72 ℃ for 40s, and reaction for 29 cycles; extension at 72 ℃ for 10min and at 4 ℃ for 10 min.

In step S103, the screening and identification of the recombinant pichia pastoris strain X33-p PICZ-LySP2 provided by the embodiment of the present invention includes:

selecting a single colony to YPD culture medium containing Zeocin +, and culturing at 29 ℃ for 12 h; adding 1mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 8000r/min for 5min, and collecting thalli; 50 μ L of ddH pH 8.0₂Dissolving DNA with O, and storing at-20 ℃ for later use; the correct positive recombinant yeast strains identified by PCR were designated X33-p PICZ-LySP2 and the empty yeast strain X33-p PICZ.

In step S103, the inducing fermentation and condition optimization of the pichia pastoris X33-p PICZ-LySP2 provided by the embodiment of the present invention include:

1) transformants identified as positive by yeast colony PCR on transformed YPD plates containing Zeocin + were picked and inoculated into 25mL BMGY liquid medium;

2) culturing at 29 deg.C and 350r/min with shaking until OD600 is 2.0; collecting part of the culture solution, centrifuging at room temperature of 6000r/min for 5min, removing supernatant, and washing thallus with sterile PBS for 2 times;

3) transferring the thallus to a 50mL BMMY culture medium, controlling the initial OD600 to be 1.0, carrying out shake culture at 29 ℃ and 350r/min for 72-96h, supplementing 1% methanol to the culture medium every 12h, sampling every 24h, analyzing the thallus growth condition, and screening high-expression strains.

The technical effects of the present invention will be further described with reference to specific embodiments.

Example 1:

materials (I) and (II)

Restriction enzymes Xho I, Xba I, Sca I, DNA Marker 15,000, DNA Marker 5000, DNA Marker 2000, 10 × Loading Buffer, rTaq enzymes were purchased from TAKARA of Japan; tris, SDS, beta-mercaptoethanol, ammonium persulfate, TEMED were purchased from Amresco, USA; zeocin antibiotics were purchased from the united states; protein Marker (180, 130, 95, 72, 55, 43, 34, 26, 17, 10) was purchased from Thermo; agarose gel DNA recovery kit, plasmid miniextraction kit, PCR product purification recovery kit, yeast genome DNA extraction kit and virus DNA/RNA extraction kit are all purchased from Beijing Tiangen Biochemical technology Co.

According to the sequence of a lyase Ly SP2 gene of a salmonella pullorum bacteriophage YSP2, a pair of primers are designed and named as Ly SP2-ZF and LySP2-ZR, restriction sites of restriction enzymes Xho I and Xba I are respectively inserted into the 5 'end and the 3' end, the primers are used for cloning the lyase LySP2 gene and constructing a eukaryotic expression vector, the theoretical amplification fragment size is 495bp, and the primer sequences are as follows:

Ly SP2-ZF:5’–ccgc|tcgagaaaagaatggctattaaaaagacaatagccg-3’

Xho I

LySP2-ZR:5’-tgct|ctagatcatttattcagatccattacacaa-3’

Xba I

second, method

1. Culture of salmonella pullorum

The Salmonella pullorum was selected by the inoculating loop and streaked on LB plate, and cultured overnight at 37 ℃. And selecting a single colony to be inoculated into an LB liquid culture medium, and performing shake culture at 37 ℃ and 160r/min until the concentration of the colony is 108 CFU/mL.

2. Culture of Salmonella phage YSP2

The host bacteria are selected and inoculated in 5mL LB liquid culture medium, and shake culture is carried out at 37 ℃ and 160r/min until OD600 is 0.6. Adding 100 mu L of phage liquid, shaking and culturing at 37 ℃ and 160r/min until the culture solution is clear and transparent. Filtering the culture solution with 0.22 μm microporous membrane, collecting filtrate, adding 30% glycerol, and subpackaging into 1.5mL centrifuge tubes, and storing at-4 deg.C and-80 deg.C respectively.

3. Extraction of Salmonella phage YSP2 genome

Respectively adding 20 mu L of protease K and 200 mu L of sample into a 1.5mL centrifuge tube, and uniformly mixing; adding 200 mu L of BB, performing vortex oscillation for 15s, and incubating for 15min at 56 ℃; adding 250 μ L of anhydrous ethanol, vortex and shake for 15s, standing at room temperature for 5 min; centrifuging at 10000r/min for 1min, and discarding waste liquid; adding 500 microliter WB, centrifuging at 10000r/min for 1min, discarding the waste liquid, and repeating once; centrifuging at 10000r/min for 1min for air drying, and transferring to a new 1.5mL RNase-free centrifuge tube; adding 20 μ L sterile water, centrifuging at room temperature 10000r/min for 1min, eluting, and collecting DNA, and storing at-20 deg.C for use.

4. PCR amplification of lyase Ly SP2 Gene

The whole genome of the phage YSP2 is used as a template, and a target gene LySP2 is amplified by PCR amplification by using a primer LySP2-ZF/LySP 2-ZR.

5. Construction and identification of expression vector p PICZ-LySP2

The yeast expression vector p PICZ-alpha A is subjected to double digestion, and the vector is connected with a LySP2 gene fragment by utilizing T4 DNA ligase. The ligation product was transformed into competent DH5 α, positive transformants were selected and plasmids were extracted for PCR and sequencing identification.

Selecting transformed single bacteria to fall into 5mL LB culture medium containing Zeocin + low salt, culturing for 6-8 hours at 37 ℃, carrying out PCR identification by taking bacteria liquid as a template to reduce false positive rate, identifying the used primers as 5' AOX on p PICZ-alpha A carrier and a target gene downstream primer LySP2-ZR, and carrying out amplification by utilizing a PCR Mix system.

The PCR amplification procedure was as follows: pre-denaturation at 94 ℃ for 10min for 1 cycle; pre-denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 35s, extension at 72 ℃ for 40s, and reaction for 29 cycles; extension at 72 ℃ for 10min and at 4 ℃ for 10 min.

Extracting plasmid, double enzyme digestion identification and 1% gel electrophoresis identification. And (3) sequencing the positive plasmid by Kuumei Biotechnology Limited, comparing and analyzing the sequencing result, and detecting whether the base mutation and the frame shift exist.

6. Screening and identification of recombinant Pichia pastoris strain X33-p PICZ-LySP2

Selecting a single colony to YPD culture medium containing Zeocin +, and culturing at 29 ℃ for 12 h; adding 1mL of bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 8000r/min for 5min, and collecting thalli; the specific operation is shown in the specification of a yeast genome extraction kit, DNA is dissolved by 50 mu L of ddH2O (pH value is 8.0), and the DNA is preserved at the temperature of-20 ℃ for later use; the correct positive recombinant yeast strains identified by PCR were designated X33-p PICZ-LySP2 and the empty yeast strain X33-p PICZ.

7. Induction fermentation and condition optimization of recombinant pichia pastoris X33-p PICZ-LySP2

Selecting transformants which are identified as positive by yeast colony PCR on the transformed YPD plate containing Zeocin +, inoculating the transformants into 25mL BMGY liquid medium (25 mL of 250mL conical flask liquid volume), culturing at 29 ℃ at 350r/min, and shaking until OD600 is 2.0; collecting part of the culture solution, centrifuging at room temperature of 6000r/min for 5min, discarding the supernatant, and washing the thallus with sterile PBS for 2 times to completely remove glycerol; transferring the thallus into a 50mL BMMY culture medium (the liquid volume of a 500mL conical flask is 50mL), controlling the initial OD600 to be 1.0, culturing at 29 ℃ under a shaking condition at 350r/min for 72-96h, supplementing 1% methanol into the culture medium every 12h to continuously induce the thallus to produce enzyme, sampling every 24h, analyzing the thallus growth condition, and screening high-expression strains.

Three, result in

1. Whole genome extraction result of phage YSP2

FIG. 2 shows the result of gel electrophoresis of the whole genome of the extracted Salmonella pullorum phage YSP 2.

2. Identification of recombinant plasmid p PICZ alpha A-LySP2

The recombinant plasmid p PICZ alpha A-LySP2 is subjected to double enzyme digestion by Xho I and Xba I, the theoretical fragment size is 3518bp and 495bp, and lane 3 of FIG. 3 shows that the band size is in line with the expectation. The recombinant plasmid was PCR amplified to obtain a theoretical fragment of 495bp, and lane 4 of FIG. 3 shows that the band size is expected.

3. Activity characterization of fermentation products

After inducing the recombinant strain X33-p PICZ-LySP2 and the control strain X33-p PICZ by shaking bottles, 100 mu L of fermentation product is added into gel holes. As shown in FIG. 4, the fermentation product of the recombinant yeast X33-pPICZ-LySP2 was effective in lysing Salmonella pullorum isolates, whereas the fermentation product of the control X33-p PICZ strain had no lysis effect.

4 the curative effect of the recombinant lyase in a pullorum disease infected chicken model

The survival rate of the chicks treated by the lyase of 60 mu g/mL and the phage of 1010PFU/mL can reach 70 percent, and the survival rate of the blank control group and the unloaded yeast expression group is lower than 30 percent.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Jilin university

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