CN111500612A

CN111500612A - Soluble porcine gastrin releasing peptide fusion protein expression vector and application thereof

Info

Publication number: CN111500612A
Application number: CN202010237006.6A
Authority: CN
Inventors: 马志禹; 郭军培; 张友文; 宗荣玲; 张淼
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-08-07

Abstract

The invention belongs to the field of molecular biological genetic engineering, relates to a soluble protein expression technology, and particularly relates to a soluble pig gastrin releasing peptide fusion protein expression vector and application thereof, wherein the soluble pig gastrin releasing peptide fusion protein expression vector pET32a (+) -pGRP gene sequence is shown as a sequence list SEQ-3. The soluble porcine gastrin releasing peptide fusion protein expression vector can improve the expression quantity of soluble pGRP and realize the soluble, stable and high-activity expression of the porcine gastrin releasing peptide.

Description

Soluble porcine gastrin releasing peptide fusion protein expression vector and application thereof

Technical Field

The invention belongs to the field of molecular biological gene engineering, relates to a soluble protein expression technology, and particularly relates to a soluble porcine gastrin-releasing peptide fusion protein expression vector and application thereof.

Background

Gastrin-releasing peptide (GRP) is a neuropeptide isolated and purified from porcine stomach tissue in 1979, and is a member of the mammalian Bombesin (bombein, BN) -like peptide family, as well as Neuromedin B (NMB). In vivo, GRPs exert a variety of important physiological functions through their receptors (GRPR), including mainly: promoting proliferation, regulating behavioral responses (e.g., stress and memory), regulating gastrointestinal hormone release, altering immune function, stimulating smooth muscle contraction in the gastrointestinal and genitourinary tracts, stimulating normal tissue and tumor growth, regulating central nervous system activities (e.g., circadian rhythm, temperature and food intake), and the like.

Since the discovery of GRPs, researchers at home and abroad have made a great deal of studies on the expression and distribution of GRPs and their receptors in vivo in animals such as humans, rats, mice, etc., and have found that GRPs and their receptors are widely expressed in the central nervous system and peripheral tissues and organs. At present, no specific antibody aiming at the pig GRP protein is found in the market, the homology of the amino acid sequence of human, rat and mouse GRP and the amino acid sequence of pig GRP is lower (68%, 70% and 68% respectively), and the antibody for detecting the human, rat and mouse GRP protein can not be used for detecting the pig GRP protein. The pig is one of the main sources of the animal protein of the main economic livestock and people in China, is also an important model animal, ensures the health of the pig, improves the production performance and the reproductive capacity of the pig, is an important responsibility of the work of animal husbandry and veterinarian, and provides an important reference value for further researching human diseases. The Open Reading Frame (ORF) of the pig GRP gene is 441bp, and 146 amino acids are coded. Therefore, the specific antibody for detecting the porcine GRP protein can be prepared, and not only can be used for researching the physiological function of the GRP in a pig body, but also can make up for the demand of the antibody in the market. However, the existing preparation method has the defects of small polypeptide synthesis amount, short storage time and the like due to the reasons of high polypeptide synthesis cost, easy degradation and the like, so that the stability and the activity of the synthesized polypeptide are unstable, and the soluble, stable and high-activity expression of the porcine gastrin releasing peptide cannot be realized by the method.

Disclosure of Invention

In order to solve the technical problems, the invention provides a soluble porcine gastrin releasing peptide fusion protein expression vector to improve the expression quantity of soluble pGRP and realize the soluble, stable and high-activity expression of the porcine gastrin releasing peptide.

The technical scheme provided by the invention is as follows:

a soluble porcine gastrin releasing peptide fusion protein expression vector, wherein the soluble porcine gastrin releasing peptide fusion protein expression vector pET32a (+) -pGRP gene sequence is shown as a sequence list SEQ-3.

The invention also provides a construction method of the soluble porcine gastrin releasing peptide fusion protein expression vector, which comprises the following steps:

constructing a recombinant plasmid pMD19T-pGRP to obtain a complete CDs sequence of the pig GRP gene with enzyme cutting sites inserted at two ends;

the target protein coding gene is cloned into an expression plasmid pET32a (+), and a recombinant protein recombinant expression vector pET32a (+) -pGRP is constructed.

Further, EcoR I and Xho I are selected as inserted enzyme cutting sites, an upstream primer 5'-GGAATTCATGCGCGGCCGCGAGT-3' and a downstream primer 5'-CCGCTCGAGTCAGTTCAGCTGGGG-3' are designed and synthesized, then, a target fragment is amplified through PCR, the target fragment is purified and recovered according to a general DNA purification and recovery kit, then, the amplified target fragment is cloned to a pMD19T vector to construct a recombinant plasmid pMD19T-pGRP, the recombinant plasmid pMD 19-pGRP is transformed into DH5 α competent cells, the recombinant plasmid pMD is subjected to inversion culture overnight at 37 ℃ on a plate containing a L B solid culture medium containing benzyl (Amp +), clone colonies are screened, single colonies are picked up to be subjected to bacterial amplification in an Amp + containing L B liquid culture medium, plasmids are extracted, and plasmid sequencing is verified.

Further, carrying out EcoR I and Xho I double digestion on pMD19T-pGRP plasmid and pET32a (+) plasmid, detecting the obtained digestion product by agarose gel electrophoresis, cutting a target band, purifying and recovering the target fragment, connecting the obtained target band under the action of T4 DNA L igase, constructing a recombinant protein expression vector pET32a (+) -pGRP, transforming the recombinant protein expression vector into DH5 α competent cells for bacterial liquid amplification, and verifying plasmid sequencing.

The invention also provides a preparation method of the soluble porcine gastrin releasing peptide fusion protein expression strain, which comprises the steps of transforming the constructed recombinant protein expression vector pET32a (+) -pGRP into B L21 (DE3) competent cells, carrying out inverted culture on a plate of L B solid culture medium containing Amp + for overnight, screening colonies, selecting single colonies to carry out bacterial amplification in L B liquid culture medium containing Amp +, and verifying whether pET32a (+) -pGRP recombinant plasmids are successfully transformed into B L21 (DE3) competent cells through bacterial liquid PCR to obtain the B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain.

The invention also provides a soluble porcine gastrin releasing peptide fusion protein expression strain, which is characterized in that the strain is a B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain and is prepared by the preparation method.

Furthermore, the bacterial strain expresses a fusion protein pGRP amino acid containing His-tag as shown in a sequence list SEQ-4.

The invention also provides a porcine gastrin releasing peptide polyclonal antibody which is prepared from B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strains.

The invention also provides a preparation method of the porcine gastrin releasing peptide polyclonal antibody, which is characterized by comprising the steps of culturing a B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain to obtain a bacterial liquid, collecting bacterial precipitates, adding a lysate, re-suspending bacteria, adding lysozyme, uniformly mixing, placing on ice, ultrasonically cracking bacteria on ice, centrifuging, collecting the bacterial lysate, purifying His-tag-pGRP fusion protein in the supernatant of the bacterial lysate, mixing the His-tag-pGRP fusion protein with an isovolumetric Freund complete adjuvant, fully emulsifying, immunizing the emulsified antigen subcutaneously on the back of a New Zealand male white rabbit by adopting a back subcutaneous multipoint injection method, anesthetizing the white rabbit after four times of immunization, sampling blood from abdominal aorta, separating serum, and purifying to obtain the porcine gastrin releasing peptide polyclonal antibody.

Further, the four immunization steps are as follows: carrying out primary immunization on the emulsified antigen under the back skin of a New Zealand male white rabbit by adopting a back subcutaneous multipoint injection method, and taking a small amount of normal serum as a negative control before immunization; after the first immunization for 14d, mixing the same pGRP fusion protein with equivalent Freund incomplete adjuvant, emulsifying, performing secondary immunization, and detecting a small amount of primary immune serum before immunization; then, 1 booster immunization is carried out every 10 days, and a small amount of serum is taken for detection before three and four immunizations.

The invention also provides application of the soluble porcine gastrin releasing peptide fusion protein expression vector in detecting porcine GRP protein.

The invention also provides a soluble porcine gastrin releasing peptide fusion protein which has immunogenicity and biological activity consistent with natural porcine GRP (pGRP).

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention discloses a soluble porcine gastrin releasing peptide fusion protein expression vector and application thereof, and the specific steps are as follows:

firstly, designing a pair of amplification primers containing enzyme cutting sites by using information biology software according to the CDs sequence of a pig GRP gene as a sequence table SEQ-1, and amplifying a target protein coding gene pig recombinant GRP gene sequence as a sequence table SEQ-2;

secondly, cloning the target protein coding gene into an expression plasmid pET32a (+), and constructing a target protein recombinant expression vector pET32a (+) -pGRP, wherein the gene sequence is shown as a sequence list SEQ-3;

thirdly, the constructed pET32a (+) -pGRP plasmid is transformed into B L21 (DE3) competent cells, IPTG induces a B L21 (DE3) recombinant protein expression strain, and fusion protein pGRP amino acid containing His-tag is expressed, wherein the fusion protein pGRP amino acid is shown as a sequence list SEQ-4;

and fourthly, carrying out ultrasonic crushing on the obtained recombinant escherichia coli thallus, and purifying the expressed soluble pGRP fusion protein by using a nickel column (His-tag protein purification).

SEQ-1 is a pig GRP gene sequence with the length of 608 bp; CDs are 68-508 sites of the sequence, and the length is 441 bp;

SEQ-2 is a pig recombinant GRP gene sequence of a target protein coding gene amplified by PCR, and the length is 457 bp; the enzyme cutting site EcoR I is the 2-7 site of the sequence; the restriction enzyme site Xho I is 449-454 site of the sequence;

SEQ-3 is a constructed fusion protein pGRP gene sequence containing His-tag, and the length is 942 bp;

SEQ-4 is a constructed His-tag-containing fusion protein pGRP amino acid sequence with the length of 313 aa;

preferably, the complete sequence of CDs is selected to be a fragment of porcine GRP of interest.

Preferably, the E.coli expression vector is pET32a (+) and the expression strain is B L21 (DE 3).

Preferably, IPTG induced fusion protein expression is performed at a concentration of 0.5mM, at a temperature of 25 ℃ and for a period of 12h, and a higher expression amount of soluble pGRP fusion protein can be obtained, and pGRP fusion protein is purified using a nickel column (His-tag protein purification).

Preferably, the recombinant pGRP fusion protein comprises the complete pGRP protein amino acid sequence.

The invention also provides the porcine gastrin-releasing peptide fusion protein prepared by the method.

The invention also provides a recombinant escherichia coli expression strain prepared by the method.

The invention also provides a preparation method of the porcine gastrin releasing peptide polyclonal antibody, which comprises the steps of adding an adjuvant into the porcine gastrin releasing peptide fusion protein, emulsifying, and immunizing experimental animals to obtain the porcine gastrin releasing peptide polyclonal antibody.

Has the advantages that:

in gene engineering expression systems such as a yeast cell expression system, an escherichia coli expression system and the like, yeast fermentation expression has the defects of easiness in degradation, instability in expression and the like, and escherichia coli expression has the characteristics of high expression level, easiness in control, low production cost and the like. Therefore, the invention aims to construct a soluble porcine gastrin releasing peptide fusion protein expression vector through an escherichia coli expression system, so as to realize soluble, stable and high-activity expression of the porcine gastrin releasing peptide.

The invention selects the escherichia coli to express the pig GRP protein, and compared with the artificial chemical synthesis of the pig GRP polypeptide, the invention has the advantages of low production cost, high expression level and easy control.

According to the invention, the solubility of the pig GRP fusion protein is greatly increased by optimizing the concentration, temperature and time of IPTG induced protein expression, the soluble pig GRP fusion protein with high expression level can be obtained, and the purified protein can be used for the research of structure and function research, antibody preparation and other methods.

The pig GRP polyclonal antibody prepared by the invention can detect pig GRP protein, makes up for the defect that no detection pig GRP protein antibody exists in the market, and provides antibody support for researching the distribution and positioning of GRP in pigs.

Drawings

FIG. 1 shows the size of the recombinant GRP gene fragment of pig;

FIG. 2 is a schematic diagram of EcoR I-pGRP-Xho I;

FIG. 3 is a map of pET32a vector;

FIG. 4 is an SDS-PAGE electrophoresis of recombinant pGRP supernatant;

FIG. 5 is an SDS-PAGE electrophoresis of the recombinant protein pGRP purification.

Detailed Description

Example 1

The following description is provided by way of specific examples, which are only intended to be a general illustration of the present invention and to facilitate a better understanding of the application of the present invention, but do not limit the scope of the present invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

Example 1, a method for constructing a soluble porcine gastrin releasing peptide fusion protein expression vector, comprising the steps of:

firstly, constructing a recombinant plasmid pMD19T-pGRP to obtain the complete CDs sequence of the pig GRP gene with enzyme cutting sites (the upstream 5 'end is EcoR I and the downstream 3' end is Xho I) inserted at both ends:

firstly, sequence analysis is carried out on CDs sequence of pig GRP gene (GenBank: MF508701.1) by using information biology software (Primer Premier 5.0), EcoR I and Xho I are selected as inserted enzyme cutting sites, upstream Primer (inserted protective base G and EcoR I enzyme cutting site GAATTC) 5'-GGAATTCATGCGCGGCCGCGAGT-3' and downstream Primer (inserted protective base CCG and Xho I enzyme cutting site CTCGAG) 5'-CCGCTCGAGTCAGTTCAGCTGGGG-3' are designed and synthesized, then, target fragment is amplified by optimized PCR reaction condition and purified and recovered according to universal type DNA purification recovery kit, then, the amplified target fragment is cloned to pMD19T vector to construct recombinant plasmid pMD 19T-RP pGRP and transformed into DH5 α competent cell, inverted culture is carried out on a plate containing L B solid culture medium containing benzyl (Amp +), colony is screened, single bacterium colony is selected to be sequenced in a sequencing liquid culture medium containing Amp + L B, bacterial amplification is carried out overnight according to high purity plasmid amplification kit, and plasmid is extracted according to small column centrifugation.

Secondly, constructing a target protein recombinant expression vector pET32a (+) -pGRP:

carrying out EcoRI and Xho I double digestion on pMD19T-pGRP plasmid and pET32a (+) plasmid according to an optimized digestion system and digestion conditions, detecting the obtained digestion product by agarose gel electrophoresis, cutting a target strip, purifying and recovering a target fragment according to a universal DNA purification and recovery kit, connecting the obtained target strip under the action of T4 DNA L igase according to an optimized connecting system, constructing a recombinant protein expression vector pET32a (+) -pGRP, transforming the recombinant protein expression vector into DH5 α competent cells, carrying out bacterial liquid amplification (the same step one as the step one for culturing, screening and amplifying the bacterial colony), and verifying the sequencing of the plasmid.

IPTG induced expression and detection of fusion protein pGRP containing His-tag

The recombinant protein expression vector pET32a (+) -pGRP constructed in the previous step is transformed into B L21 (DE3) competent cells, inverted and cultured overnight at 37 ℃ on a plate of L B solid culture medium containing Amp +, colonies are screened, single colonies are picked out to be subjected to bacterial amplification in L B liquid culture medium containing Amp +, and bacterial liquid PCR is used for verifying whether pET32a (+) -pGRP recombinant plasmids are successfully transformed into B L21 (DE3) competent cells, so that a B L21 (DE3) recombinant protein expression strain is obtained.

Culturing the obtained B L (DE3) recombinant protein expression strain at a constant temperature of 37 ℃ for 6-8h (preferably, the OD600 value of the bacterial liquid is 0.6-0.8), adding IPTG (isopropyl thiogalactoside) into the bacterial liquid to induce the expression of the recombinant protein, wherein the IPTG (isopropyl thiogalactoside) is used as an active inducing substance of β -galactosidase in the bacteria and can induce the expression of the recombinant protein in the B L (DE3) recombinant protein expression strain under a certain condition, carrying out induced expression under an optimized IPTG inducing condition (the IPTG inducing concentration is 0.5mM, the inducing temperature is 25 ℃ and the inducing time is 12h), then collecting the bacterial liquid, centrifugally collecting the bacterial cells, carrying out resuspension on the collected bacterial cells by PBS solution, carrying out centrifugation again to obtain the bacterial cells, adding a non-deformable liquid and fully resuspension, adding lysozyme to 1mg/m, gently mixing, placing the bacterial cells on ice for fully cracking the bacteria, carrying out ultrasonication on the bacteria by centrifugation, obtaining a supernatant, taking the supernatant of the bacterial cells, carrying out electrophoresis on 5 SDS, carrying out boiling on the mixed supernatant, carrying out decoloration on the mixed protein, carrying out electrophoresis on the stained gel on the supernatant of the supernatant, carrying out electrophoresis on the stained protein, carrying out electrophoresis on the stained gel.

The fusion protein pGRP is recombinant protein with 6 His-tag at the N end, 314 amino acids in total and 33.98kDa in molecular weight.

Example 2 preparation of polyclonal antibodies to porcine gastrin-releasing peptide, the procedure was as follows:

induced expression and purification of His-tag-pGRP fusion protein

Escherichia coli was cultured using the B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain obtained in example 1, then, bacterial liquid of 500m L was obtained according to the IPTG induction conditions optimized in example 1 (induction concentration of 0.5mM, induction temperature of 25 ℃ and induction time of 12h), bacterial pellet was collected, lysate was added at a ratio of 2-5m L non-denaturing lysate (containing an appropriate amount of protease inhibitor mixture) per gram of wet weight of bacteria, bacterial suspension was resuspended, lysozyme was added to a concentration of 1mg/m L, and the mixture was left on ice for 30 min.

Beyogold produced by Biyun sky^TMHis-tag Purification Resin, i.e., a nickel column (His-tag protein Purification medium) purified His-tag-pGRP fusion protein in the supernatant of the above bacterial lysate. Taking a proper amount of purified solution for denaturation, and detecting the purity of the purified fusion protein pGRP by 10% SDS-PAGE gel electrophoresis; western blot hybridization identification is carried out on the His monoclonal antibody and the purified fusion protein pGRP, and the protein obtained after induction expression and purification is proved to be the fusion pGRP protein with His-tag and can be used as an antigen for preparing a follow-up pig GRP polyclonal antibody.

Preparation of pGRP polyclonal antibody

The method comprises the steps of utilizing a BCA protein concentration determination kit to perform concentration determination on purified fusion protein pGRP, taking a proper amount of pGRP fusion protein to be mixed with equal volume of Freund's complete adjuvant (CFA), fully emulsifying, performing primary immunization on emulsified antigens under the back skin of New Zealand male white rabbits by adopting a back subcutaneous multipoint injection method, taking a small amount of normal serum as a negative control before immunization, mixing the same pGRP fusion protein with equal volume of Freund's incomplete adjuvant (IFA) after 14d of primary immunization, performing secondary immunization after emulsification, taking a small amount of primary immune serum before immunization for detection, performing booster immunization for 1 time every 10d, respectively taking a small amount of serum before three times of immunization and four times of immunization, detecting the titer of antibodies by adopting an indirect E L ISA method, determining whether animals are immunized again according to the titer of the antibodies, anesthetizing the white rabbits after 10d of four times of immunization, collecting blood from abdominal aorta, separating, keeping at-80 ℃ for later use, purifying antibodies by adopting a BCA protein purification kit, and performing Western Blot specific detection on Blot antibodies and blood serum specificity detection by adopting a histochemical reaction method.

Sequence listing

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ctttaaagac atgggtccaa gactcaaagt tgatggactc tctgctccag gttctcagca 480

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cccatatgct tatgaaggag gaaacatgaa agaacagctg agggaataca ttcggtggga 240

agacgccaca aggaatttgc tgagcctcct agaagcaaag gggattggaa gccatcagcc 300

ccctcaatgg gagcccctgg gcattcgtca gtctacttgg gattcaaagg atggcagcaa 360

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Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

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Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

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Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

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Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

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Ala Asp Ile Gly Ser Glu Phe Met Arg Gly Arg Glu Phe Pro Leu Val

165 170 175

Leu Leu Ala Leu Val Leu Cys Gln Ala Pro Arg Gly Pro Ala Ala Pro

180 185 190

Val Ser Val Gly Gly Gly Thr Val Leu Ala Lys Met Tyr Pro Arg Gly

195 200 205

Asn His Trp Ala Val Gly His Leu Met Gly Lys Lys Ser Thr Gly Glu

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Ser Pro Tyr Ala Tyr Glu Gly Gly Asn Met Lys Glu Gln Leu Arg Glu

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Tyr Ile Arg Trp Glu Asp Ala Thr Arg Asn Leu Leu Ser Leu Leu Glu

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Ala Lys Gly Ile Gly Ser His Gln Pro Pro Gln Trp Glu Pro Leu Gly

260 265 270

Ile Arg Gln Ser Thr Trp Asp Ser Lys Asp Gly Ser Asn Phe Lys Asp

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Met Gly Pro Arg Leu Lys Val Asp Gly Leu Ser Ala Pro Gly Ser Gln

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His Glu Gly Arg Ile Pro Gln Leu Asn

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Claims

1. The soluble porcine gastrin releasing peptide fusion protein expression vector is characterized in that the soluble porcine gastrin releasing peptide fusion protein expression vector pET32a (+) -pGRP gene sequence is shown as a sequence list SEQ-3.

2. The method of constructing the soluble porcine gastrin-releasing peptide fusion protein expression vector of claim 1, comprising the steps of:

3. The method for constructing the soluble porcine gastrin releasing peptide fusion protein expression vector of claim 2, is characterized in that EcoR I and Xho I are selected as inserted enzyme cutting sites, an upstream primer 5'-GGAATTCATGCGCGGCCGCGAGT-3' and a downstream primer 5'-CCGCTCGAGTCAGTTCAGCTGGGG-3' are designed and synthesized, then, a target fragment is amplified by PCR and purified and recovered according to a universal DNA purification and recovery kit, then, the amplified target fragment is cloned to a pMD19T vector to construct a recombinant plasmid pMD19T-pGRP, the recombinant plasmid pMD 19-pGRP is transformed into DH5 α competent cells, the recombinant plasmid pMD is inversely cultured on a plate of a L B solid culture medium containing aminobenzyl (Amp +) at 37 ℃ overnight, clone colonies are screened, and single colonies are picked up to be subjected to bacterial amplification in an L B liquid culture medium containing Amp +, extracted and plasmid sequencing is verified.

4. The method for constructing the soluble porcine gastrin releasing peptide fusion protein expression vector of claim 2, wherein EcoR I and Xho I double enzyme digestion is carried out on pMD19T-pGRP plasmid and pET32a (+) plasmid, the obtained enzyme digestion product is detected by agarose gel electrophoresis, a target strip is cut, a target fragment is purified and recovered, the obtained target strip is connected under the action of T4 DNA L igase, a recombinant protein expression vector pET32a (+) -pGRP is constructed and is transformed into DH5 α competent cells for bacterial liquid amplification, and plasmid sequencing is verified.

5. A method for preparing a soluble porcine gastrin releasing peptide fusion protein expression strain is characterized in that the soluble porcine gastrin releasing peptide fusion protein expression vector of claim 1 is transformed into B L21 (DE3) competent cells, the soluble porcine gastrin releasing peptide fusion protein expression vector is subjected to inverted culture on a flat plate of an Amp + containing L B solid culture medium for overnight, colonies are screened, single colonies are selected to be subjected to bacterial amplification in an Amp + containing L B liquid culture medium, whether pET32a (+) -pGRP recombinant plasmids are successfully transformed into B L21 (DE3) competent cells is verified through bacterial liquid PCR, and the B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain is obtained.

6. A soluble porcine gastrin releasing peptide fusion protein expression strain, which is a B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain and is prepared by the preparation method of claim 5.

7. The soluble porcine gastrin-releasing peptide fusion protein expression strain of claim 6, wherein the strain expresses the His-tag containing fusion protein pGRP amino acids as shown in the sequence list SEQ-4.

8. A polyclonal antibody against porcine gastrin-releasing peptide, which is prepared from the recombinant protein expression strain B L21 (DE3) -pET32a (+) -pGRP of claim 6.

9. A preparation method of a porcine gastrin releasing peptide polyclonal antibody is characterized in that B L21 (DE3) -pET32a (+) -pGRP recombinant protein expression strain of claim 6 is cultured by escherichia coli to obtain a bacterial liquid, the bacterial liquid is collected, a bacterial precipitate is added, a lysate is added, the bacterial liquid is resuspended, lysozyme is added, the lysozyme is mixed uniformly and placed on ice, then, the bacteria are ultrasonically cracked on ice, the bacterial lysate is collected after centrifugation, His-tag-pGRP fusion protein in the supernatant of the bacterial lysate is purified, the His-tag-pGRP fusion protein is mixed with an isovolumetric Freund complete adjuvant and is fully emulsified, the emulsified antigen after emulsification is immunized subcutaneously at the back of a New Zealand male white rabbit by adopting a subcutaneous multipoint injection method, after four times of immunization, the white rabbit is anesthetized, the abdominal aorta is subjected to blood sampling, serum is separated, and the porcine gastrin releasing peptide polyclonal antibody is obtained after purification.

10. Use of the soluble porcine gastrin-releasing peptide fusion protein expression vector of claim 1 for detecting porcine GRP protein.