CN108129570B

CN108129570B - Preparation and application of fusion bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation

Info

Publication number: CN108129570B
Application number: CN201711421449.5A
Authority: CN
Inventors: 高荣; 万小平; 肖永乐; 马常俊; 黎淩; 朱玉华; 胡立博; 刘建华; 田玉虎; 王泽洲; 吕学斌; 李江淩
Original assignee: Shenzhen Qianhai Jinzhuo Biotechnology Co ltd; Sichuan University
Current assignee: Sichuan Sanyoukang Biotechnology Co ltd; Sichuan University
Priority date: 2017-12-25
Filing date: 2017-12-25
Publication date: 2020-09-25
Anticipated expiration: 2037-12-25
Also published as: CN108129570A

Abstract

The invention discloses preparation and application of an antibacterial peptide fusion cytokine FBAPIL2 co-expression biological agent. The antibacterial peptide fusion cytokine FBAPIL2 provided by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is protein of sequence 1 in a sequence table; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 1 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that the FBAPIL2 can promote the proliferation of lymphocytes, erythrocytes and leukocytes, inhibit the growth of pathogenic microorganisms, promote the secretion of nonspecific antibodies (IgG, IgG1 and IgG2a) and disease specific antibodies, and improve the immunocompetence and survival rate of animals.

Description

Preparation and application of fusion bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation

Technical Field

The invention belongs to the technical field of biology, and particularly relates to preparation and application of a fusion bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation.

Background

The breeding industry (animal husbandry and aquatic products) is an important component of the large agriculture, is one of the important roots of agricultural modernization in China and rural economic development and social stability, is developed in advanced countries in Europe and America, and accounts for more than 50 percent of the proportion of the large agriculture. It provides high-grade food such as meat, milk, eggs, fur and the like and important raw materials for light textiles for human beings, and livestock products are main sources of nutrient substances such as high-quality protein, mineral substances, vitamins and the like for people, and the safety of the livestock products is directly related to the body health of human beings. In recent years, malignant events such as poisoning, disease infection and the like caused by livestock products, such as mad cow disease in Europe, foot and mouth disease, avian influenza in Asia and the like, have great influence on livestock production and food processing industry. Animal epidemic diseases become important factors influencing the safety of livestock products, the health of human bodies and the social stability and increasing income and enriching of vast farmers and herders. In the breeding industry of China, more than 30 important animal infectious diseases are available; at present, not only old diseases are not eliminated, but also new diseases are continuously emerged in China, the average morbidity and mortality of animal infectious diseases are as high as about 15-20% and 8-10%, and at least 400 billion yuan of direct economic loss is caused to the breeding industry in China every year. Modern humans are facing a serious challenge where infection by pathogenic microorganisms is difficult to control. Therefore, the high-efficiency prevention and treatment of infectious diseases and the promotion of the healthy growth of animals are the core for ensuring the improvement of economic benefits and sustainable development of the breeding industry in China, and are also important technical problems which are urgently needed to be solved for protecting the food sanitation and the life health of people; and moreover, the animal husbandry in China faces a large market in the world, breaks through the green barrier, participates in international market competition, and inevitably selects new images.

The breeding industry is the main support for the agricultural and rural economic development of China at present, the marketing amount of pigs in China in 2014 reaches 7 hundred million and more, and the pork yield reaches 5670 more than ten thousand tons; the production value of the Sichuan aquaculture accounts for about 54 percent of the total agricultural value, and has great influence on the income of farmers and the development of rural areas. The feed industry which is one of the most important foundations of the breeding industry is also greatly improved, the yield of the compound feed for pigs in 2014 in China currently reaches 8456 ten thousand tons, more than 3000 ten thousand tons of concentrated feed are obtained, and 1000 ten thousand tons of additive premix feed are obtained. The feed becomes the second major producing country in the world, the feed industry in Sichuan develops rapidly, the total yield of the feed in the whole province in 2013 reaches 1067.90 ten thousand tons, wherein the total yield of the feed for pigs reaches 656.62 ten thousand tons and accounts for 61.49 percent; 312.16 ten thousand tons of poultry feed accounting for 29.23 percent; the aquatic feed accounts for 79.64 ten thousand tons, and accounts for 7.46 percent.

The main problems which limit the further development of the breeding industry in China and improve the economic benefit are the control of animal diseases, the improvement of the quality of animal products and the development and production of high-quality cheap feeds. The export animal products in China account for very low proportion of export due to lower quality of quarantine and medicine residue, for example, more than 23 ten thousand tons of pork account for only about 4 percent of international trade volume. Especially 90% of protein raw materials such as bean pulp of Sichuan animal feed and 70% of energy feed raw materials such as corn and the like depend on external supply, which seriously restricts the production development of animal husbandry and the improvement of economic benefit in our province. Therefore, how to develop high-efficiency special biological feed by using high-new biotechnology, fully utilize local feed resources, reduce the use of antibiotic additives in the feed, overcome the drug residue harm of animal products, guarantee the production of green organic animal food, and protect the life health and safety of people becomes an important scientific and technological and social economic problem which needs to be solved urgently.

At present, the livestock and poultry breeding industry in China is gradually enlarged along with the improvement of the intensification degree, the livestock and poultry breeding scale and the breeding density are forced to control the pressure of the spread and spread of more than 30 infectious pathogen infections, particularly the successive cyclic outbreak of severe diseases such as avian influenza and respiratory reproduction syndrome, and the like, so that the abuse condition of drug additives such as antibiotics and the like in feed is very serious, and the public hazard problems of livestock and poultry diseases and antibiotic feed additives become bottlenecks which restrict the development level and the economic benefit improvement of livestock and poultry breeding industry in China. The abuse of the feed antibiotic additive not only increases the feeding cost, but also obviously enhances the drug resistance and the pathogenicity of pathogenic microorganisms, and the health level of the livestock body is weakened due to the toxic and side effects of the drugs, so that the immunity and the disease resistance are obviously reduced. In such a vicious circle, various virulent infectious diseases of livestock and poultry frequently occur and are difficult to control and treat. On the other hand, the drug residue and the increasingly serious food safety problem of the livestock and poultry products not only directly threaten the health of human beings, but also hinder the development of the breeding industry.

The addition of antibiotics to animal feed, long-term or improper use of which not only induces drug resistance of pathogens, but also causes drug residues in animal products, which causes serious food safety hazards, damages to human health and incurs drug failure for the treatment of antibiotics. Developed regions such as the european union and the united states began to restrict antibiotics as feed additives comprehensively in 2006. China will also severely restrict the feed addition of antibiotics. The modern breeding industry urgently needs to develop novel biological feed and additives thereof which replace the traditional antibiotics, have no residue, no resistance induction, safety and no pollution.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the immunity of animals.

In order to solve the technical problems, the invention provides a protein which comprises bovine antibacterial peptide and interleukin 2.

The protein is any one of the following proteins a) to e):

a) the amino acid sequence comprises the protein of the amino acid sequence shown in the sequence 1 in the sequence table;

b) the amino acid sequence consists of amino acid residues shown in a sequence 1 in a sequence table;

c) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined by a) or b) and has the function of improving the animal immunity;

d) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in a) or b) and having a function of enhancing the animal's immunity;

e) a fusion protein obtained by connecting a label to the N-terminal and/or the C-terminal of the protein defined in any one of a) to d).

Nucleic acid molecules encoding the above proteins are also within the scope of the present invention.

The nucleic acid molecule is a nucleic acid molecule represented by any one of the following 1) to 4):

1) the coding sequence comprises a sequence 2 in a sequence table;

2) the coding sequence is sequence 2 in the sequence table;

3) DNA molecules which hybridize under stringent conditions with the DNA molecules defined in 1) or 2) and which code for the proteins mentioned above;

4) a DNA molecule which has more than 80% or more than 90% of homology with the DNA molecule defined in 1) or 2) and codes the protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 2 encodes FBAPIL2 shown in the sequence 1.

The nucleotide sequence of FBAPIL2 of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of FBAPIL2 isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode FBAPIL2 and have FBAPIL2 function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions are hybridization and membrane washing 2 times at 68 ℃ for 5min in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing 2 times at 68 ℃ for 15min in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

Any of the following biomaterials 1) -3) is also within the scope of the present invention:

1) an expression cassette comprising the nucleic acid molecule;

2) a recombinant vector comprising the nucleic acid molecule;

3) recombinant bacteria or transgenic cell lines containing the nucleic acid molecules;

4) and (3) a fermentation product of the recombinant bacterium.

In the above applications, the expression cassette containing a nucleic acid molecule encoding FBAPIL2 (FBAPIL2 gene expression cassette) described in B2) refers to a DNA capable of expressing FBAPIL2 in a host cell, and the DNA may include not only a promoter for promoting transcription of FBAPIL2 gene, but also a terminator for terminating transcription of FBAPIL2 gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the FBAPIL2 gene expression cassette can be constructed by using an existing vector.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may specifically be a pGAPZ α a vector.

B3) The recombinant vector may contain a DNA sequence shown in sequence 2 for encoding FBAPIL 2. Further, the recombinant vector may specifically be pG-P. pG-P is a recombinant vector obtained by replacing a DNA fragment between EcoR I and Xba I recognition sequences of a pGAPZ alpha A vector with an FBAPIL2 gene shown in a sequence 1.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the yeast is Pichia pastoris SMD 1168.

In the above-mentioned application, the recombinant microorganism may be one obtained by introducing an expression cassette containing a nucleic acid molecule encoding FBAPIL2 into a yeast. The recombinant microorganism may be specifically a recombinant microorganism obtained by introducing a recombinant vector containing an FBAPIL2 expression cassette into yeast. When the recombinant vector is introduced into the yeast, the recombinant vector may be directly introduced into the yeast, or the recombinant vector may be linearized and then introduced into the yeast. The yeast can be pichia pastoris SMD 1168.

In one embodiment of the present invention, the recombinant microorganism is a recombinant microorganism obtained by introducing the pG-P into Pichia pastoris SMD 1168. The preparation method of the recombinant microorganism specifically comprises the following steps: linearizing the pG-P to obtain linearized pG-P; and introducing the linearized pG-P into pichia pastoris SMD1168 to obtain a recombinant microorganism, wherein the name of the recombinant microorganism is SMDpG-P.

In the above application, the fermentation product of the recombinant microorganism can be prepared according to a method comprising the following steps: culturing the recombinant microorganism to express the coding gene of FBAPIL2 to obtain a fermentation product of the recombinant microorganism.

In the above applications, the transgenic cell line does not comprise propagation material.

The application of the above protein or the above nucleic acid molecule or the above biological material in the following C1 or C2 is also within the scope of the present invention:

c1, improving animal immunity;

c2, preparing the product for improving the animal immunity.

In the application, the animal immunity improving effect is at least one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, increasing animal development and growth weight gain;

and/or the pathogenic microorganism is in particular escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus or classical swine fever virus;

and/or, the immune cell is in particular a lymphocyte, a red blood cell or a white blood cell;

and/or, the antibody is specifically an IgG, IgG1 and/or IgG2 a.

The following products X1 or X2 are also within the scope of the invention:

x1, a biological agent comprising the following X3a, X3b or X3 c:

x3a, the above protein;

x3b, the above nucleic acid molecule;

x3c, the biomaterial described above;

x2, kit for improving animal immunity, which consists of the X1 and antibiotics.

In the product, the biological agent can be X3a, X3b or X3c as an active ingredient, and can also be a composition which combines X3a, X3b or X3c with other substances capable of improving the animal immunity as an active ingredient.

Or, a method for enhancing the immunocompetence of an animal, comprising administering to the animal the above protein or the above nucleic acid molecule or the above biological material or the above biological agent or the kit, thereby enhancing the immunocompetence of the animal.

In the above, the animal is any one of H1-H3:

h1, mammalian;

h2, pig;

h3, mouse.

In the above product, the antibiotic may be ampicillin and/or kanamycin.

In order to solve the above technical problems, the present invention also provides a method for enhancing the immunocompetence of an animal, which may comprise administering FBAPIL2, the biological material or the biological agent to the animal, thereby enhancing the immunocompetence of the animal.

In the invention, the animal immunity improving capability can be any one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, improving animal development and growth weight gain.

The pathogenic microorganism can be Escherichia coli, Staphylococcus aureus, Mycoplasma hyopneumoniae, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) or classical swine fever virus (HCV or CSFV), such as Escherichia coli Standard (G)^-) Escherichia coli resistant bacterium (G)^-) Staphylococcus aureus (G)⁺) Staphylococcus aureus resistant bacterium (G)⁺)。

The immune cell may be a lymphocyte (e.g., CD4+ or CD8+), a red blood cell, or a white blood cell.

The antibody may be IgG, IgG1, and/or IgG2 a. The antibody may specifically be an antibody of the pathogenic microorganism.

The vaccine may in particular be a vaccine against the pathogenic microorganism.

In the present invention, the improvement of the animal's immunity may specifically be an improvement of the animal's immunity to the pathogenic microorganism, such as an improvement of the immunity to mycoplasma hyopneumoniae which causes swine enzootic pneumonia (MP).

In the present invention, the animal may be a mammal. The mammal may be specifically a pig or a mouse.

In the invention, the products for improving the animal immunity can be medicines for improving the animal immunity.

Experiments prove that the co-expression FBAPIL2 molecule and the fermentation product of the recombinant yeast containing the FBAPIL2 gene have the following functions: promoting the increase of lymphocytes, erythrocytes and leukocytes of animals, and increasing the content of lymphocytes, erythrocytes and leukocytes in the animals administered with FBAPIL2 by more than 10%; obviously inhibit the growth of pathogenic microorganisms, and the amount of the pathogenic microorganisms applied with FBAPIL2 can be reduced by more than 20 percent; promote the secretion of nonspecific antibody (IgG, IgG1, IgG2a) and disease specific antibody, and increase the content of nonspecific antibody and disease specific antibody in animal body administered with FBAPIL2 by 40% -60%; the expression of immune related genes is promoted, so that the immune anti-infection capability of animals is improved, the toxic attack survival rate of the animals taking the FBAPIL2 is obviously improved by at least 60 percent, and the growth and weight gain rate is more than 10 percent.

Drawings

FIG. 1 shows the RT-PCR results for the gene of interest in SMDpG-2B.

FIG. 2 shows the results of protein expression level measurements of the target gene in SMDpG-2B.

FIG. 3 is a graph showing the effect of yeast strain SMDpG-2B fermentation supernatant on porcine lymphoblastoid cell proliferation.

FIG. 4 shows the inhibitory effect of SG2B on Staphylococcus aureus standard bacteria.

FIG. 5 shows the inhibitory effect of SG2B on drug-resistant bacteria of Staphylococcus aureus.

FIG. 6 shows the inhibitory effect of SG2B on E.coli standard bacteria.

FIG. 7 shows the inhibitory effect of SG2B on E.coli-resistant bacteria.

FIG. 8 shows the time course of peripheral blood leukocytes of mice under different treatments.

FIG. 9 shows the change of CD4+ lymphocytes per 10000 cells in peripheral blood of mice.

FIG. 10 shows the change of CD8+ lymphocytes per 10000 cells in peripheral blood of mice.

FIG. 11 shows the change of IgG, a non-specific antibody, in peripheral blood of mice of different groups.

FIG. 12 shows the variation of IgG1, a non-specific antibody in peripheral blood, among different groups of mice.

FIG. 13 shows the change of IgG2a, a non-specific antibody in peripheral blood, among mice of different groups.

FIG. 14 shows the content of MP-specific antibodies in peripheral blood of mice of different groups.

FIG. 15 shows the expression level of the Th1 type cytokine TNF-alpha gene in peripheral blood of different groups of mice.

FIG. 16 shows the expression level of Th2 type cytokine IL4 gene in peripheral blood of different groups of mice.

FIG. 17 shows the expression levels of TLR gene in peripheral blood of different groups of mice.

FIG. 18 shows the expression levels of peripheral blood immunological memory related factors of mice of different groups.

FIG. 19 shows survival rates of different groups of mice after challenge experiments with E.coli.

Fig. 20 shows net weight gain of piglets in each group.

FIG. 21 is the dynamic change of leukocytes during piglet growth.

FIG. 22 is a graph of the dynamic change of CD8+ T lymphocytes in peripheral blood during piglet growth.

FIG. 23 is a graph showing the dynamic change of CSF-specific antibodies in peripheral blood during piglet growth.

Fig. 24 is a graph showing the dynamic changes of PRRSV-specific antibodies in peripheral blood during piglet growth.

FIG. 25 shows the expression level of peripheral blood TLR gene during the growth period of piglets.

FIG. 26 shows the expression level of peripheral blood immunological memory related factor gene during the growth period of piglets.

FIG. 27 shows the expression level of the peripheral blood signal transduction molecule Bcl-2 gene during the growth of piglets.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The pGAPZ. alpha.A vector in the examples described below is Invitrogen, catalog number V20020.

Pichia pastoris SMD1168 in the examples below is Invitrogen, catalog number C17500.

The Changbai pig in the following embodiment is a product of Jianyang base of the performance testing center of Sichuan province breeding pigs.

The Tibetan pigs in the following examples are products of Jianyang base of the performance testing center of Sichuan province breeding pigs.

Escherichia coli Standard bacteria (G) in the following examples^-) Is the ATCC (American Type CultureCollection) product, catalog number 25922.

Escherichia coli-resistant bacteria (G) in the following examples^-) The biological material is provided for animal epidemic prevention and control and food safety of Sichuan university in key laboratories of Sichuan province, and the public can obtain the biological material from the applicant, and the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

Staphylococcus aureus Standard bacteria (G) in the following examples⁺) Is the ATCC (American Type CultureCollection) product, catalog number 29213.

Staphylococcus aureus resistant bacteria (G) in the following examples⁺) The biological material is provided for animal epidemic prevention and control and food safety of Sichuan university in key laboratories of Sichuan province, and the public can obtain the biological material from the applicant, and the biological material is only used for repeating relevant experiments of the invention and cannot be used for other purposes.

The female ICR mice in the following examples are products of the institute for laboratory animals in the national Hospital, Sichuan province.

Example 1 bovine antimicrobial peptide Interleukin 2 fusion protein (FBAPIL2) and Gene encoding same

First, fusion protein FBAPIL2 and obtaining of coding gene thereof

The bovine antibacterial peptide and interleukin 2 are fused to obtain fusion protein which is named as FBAPIL 2.

The amino acid sequence of the fusion protein is shown as sequence 1 in the sequence table, the fusion gene for coding the fusion protein FBAPIL2 is named as FBAPIL2, and the nucleotide sequence of the fusion gene is sequence 2.

Wherein, the 265 rd-353 th site of the sequence 1 is bovine antibacterial peptide, the 155 th-264 th site is connecting peptide, and the 1 st-154 th site is interleukin 2;

the 793-1059 position of the sequence 2 is a bovine antibacterial peptide encoding nucleic acid, the 463-792 position is a connecting peptide encoding nucleic acid, and the 1-462 positions are interleukin 2 encoding nucleic acids.

The protein can be obtained by artificially synthesizing coding nucleic acid and performing prokaryotic expression.

Secondly, preparation of recombinant vector for expressing fusion protein FBAPIL2

The recombinant vector pGAPZ alpha A-2B (pG-2B for short) is obtained by replacing a DNA fragment between EcoR I and Xba I recognition sequences of the pGAPZ alpha A vector with FBAPIL2 gene shown in sequence 2 and keeping other sequences of the vector unchanged, and expresses bovine antibacterial peptide interleukin 2 fusion protein (FBAPIL2) shown in sequence 1.

Preparation of recombinant bacteria for expressing fusion protein FBAPIL2

1. Preparation of recombinant bacteria

Digesting pG-2B with AvrII enzyme to obtain linearized pG-2B; and (3) introducing the linearized pG-2B into pichia pastoris SMD1168 to obtain recombinant yeast (sequencing a PCR product by adopting bacterial liquid PCR identification and RT-PCR), and naming the recombinant yeast as SMDpG-2B.

The primer sequence for PCR identification of the bacterial liquid is F: 5'-ATGTATAAGATGCAGCTCTTGT-3' and R:

5'-CTAATGGTGATGGTGATGAT-3', positive for amplified fragments;

the primer sequence identified by RT-PCR is F: 5'-ATGTATAAGATGCAGCTCTTGT-3' and R:

5'-CTAATGGTGATGGTGATGAT-3', positive amplified fragments.

The same method is adopted, and the pGAPZ alpha A is digested by the AvrII enzyme to obtain the linearized pGAPZ alpha A; and (3) introducing the linearized pGAPZ alpha A into pichia pastoris SMD1168 to obtain a control recombinant strain SMDpG.

2. Detection of recombinant bacteria

1) Expression detection at RNA level

The recombinant strain SMDpG-2B was inoculated into YPD medium containing 100. mu.g/ml Zeocin, cultured overnight at 28 ℃ and 200rpm, and centrifuged to obtain SMDpG-2B cells. The total RNA of the SMDpG-2B cells was extracted, and the expression level of the target gene in SMDpG-2B was determined by using a primer set (F: 5'-ATGTATAAGATGCAGCTCTTGT-3' and R: 5'-CTAATGGTGATGGTGATGAT-3').

The results are shown in FIG. 1, Lane 1-2: interleukin-antimicrobial peptide amplification; m is Trans 2K plus DNAmarker; the target band in SMDpG-2B indicates that the fusion gene FBAPIL2 in SMDpG-2B is expressed at RNA level.

The control recombinant strain SMDpG was tested in the same manner without the expression of the fusion gene FBAPIL 2.

2) Protein expression level detection

The recombinant strain SMDpG-2B (SG2B) is inoculated in YPD medium containing 100 mu g/ml Zeocin for fermentation culture at 28 ℃ and 200rpm, fermentation liquid is taken out at different times of fermentation respectively for centrifugation, supernatant liquid is taken out, and fusion protein in the supernatant liquid is detected by an ELISA kit of HIS-Tag (all antibody detection reagents are in the supernatant liquid). The supernatant obtained by fermentation with the control recombinant Pichia pastoris SMD1168 was used as a negative control (the control recombinant strain SMDpG is a control, and the result is shown in FIG. 2, and the OD thereof is₄₅₀<0.02, much lower than SG2B fermentation supernatant).

As shown in FIG. 2, compared with Pichia pastoris SMD1168, the recombinant strain SMDpG-2B (SG2B) produces the target fusion protein FBAPIL2, and the expression level is the highest in 72 hours of fermentation.

The result shows that the recombinant strain SMDpG-2B expresses the target fusion protein FBAPIL 2.

Example 2 Effect of bovine antimicrobial peptide Interleukin 2 fusion protein (FBAPIL2) on lymphocyte proliferation

1. Preparation of recombinant strain SMDpG-2B fermentation supernatant

(1) The recombinant strain SMDpG-2B (hereinafter referred to as SG2B) obtained in example 1 was inoculated into 3mL of medium 1 (medium 1 was a liquid medium obtained by adding bleomycin (Zeocin) to YPD medium, and the concentration of bleomycin was 100mg/mL), and cultured at 28 ℃ and 200rpm for overnight activation of the strain.

(2) Taking 300 mu L of the bacterial liquid obtained in the step (1), inoculating the bacterial liquid into a 100mL triangular flask containing 30mL YPD medium, performing 28 ℃,shaking table fermentation at 200rpm for 48h (OD)₆₀₀Is 25).

(3) 5mL of the bacterial suspension obtained in step (2) was centrifuged at 12000 Xg for 2min, and the resulting supernatant was named SG2B fermentation supernatant.

2. Protease treatment recombinant bacterium SMDpG-2B fermentation supernatant

Measuring the pH of the SG2B fermentation supernatant obtained in the step 1 by using a pH test paper, respectively adjusting the optimum action pH value of the SG2B fermentation supernatant to 7.0 of Trypsin and the optimum action pH value of pepsin to 2.0 by using 2mol/L NaOH and 1 mol/L HCl, respectively adding a Trypsin solution (Beijing Sorbey Technology Co., Ltd., Trypsin-EDTA digestive juice (Trypsin-EDTA) containing 0.25% of Trypsin and 0.02% of EDTA, catalog number: 9002-07-7) and a pepsin solution (Beijing Sorbey Technology Co., Ltd., 0.1% aqueous solution pH 4.0, enzyme activity: 3000. about 3500NFU/g, catalog number: 9001-75-6) to simulate the action of digestive enzyme for degrading protein, so that the final enzyme concentration is 0.5mg/mL, carrying out water bath enzyme reaction at 37 ℃ for 1h to obtain the SG2B fermentation supernatant and the SG2B fermentation supernatant treated by Trypsin, and storing in a refrigerator at-20 deg.C.

According to the method, the SMDpG-2B is replaced by SMDpG (hereinafter referred to as SG) and other steps are not changed, SG fermentation supernatant, trypsin-treated SG fermentation supernatant and pepsin-treated SG fermentation supernatant are obtained respectively and are stored in a refrigerator at the temperature of minus 20 ℃ for later use.

3. Effect of the fusion protein FBAPIL2 on lymphocyte proliferation

1) Preparation of porcine lymphocytes

Under the aseptic condition, 5mL of peripheral blood of the forechamber vein of the long and white pig is collected by a blood collection tube (containing EDTA-2K anticoagulant), and the lymphocytes of the long and white pig are separated according to the operation steps of a pig lymphocyte separation solution (preheating the separation solution at 37 ℃ before use, fully shaking and uniformly mixing).

2) Detection of biological activity of SMDpG-2B fermentation product in vitro

(1) After culturing the porcine lymphocytes separated in the step 1) for 24h, transferring the porcine reproductive and respiratory syndrome cells in the culture dish into a clean 15mL sterile centrifuge tube, and centrifuging at 1500rpm at room temperature for 15min to collect cell bodies.

(2) The cells were washed with RPMI1640 complete medium (containing the penicillin-streptomycin double antibody, 10% fetal bovine serum) and repeated 2 times, and centrifuged at 1500rpm for 15min at room temperature to collect the cell pellet.

(3) Cells were selected and adjusted to about 6 × 10 cells by complete culture in RPMI1640 containing 20mg/mL α -MM⁶Cell suspension was obtained at one/mL.

(4) To each well of the 96-well cell plate, 75. mu.L of the cell suspension of step (3), 45. mu.L of the sample solution, and 30. mu.L of RPMI1640 complete medium containing 20mg/mL of α -MM (methyl mannoside) were added according to the layout.

Wherein the sample liquid is SG2B fermentation supernatant obtained in the

steps

1 and 2, SG2B fermentation supernatant processed by trypsin, SG2B fermentation supernatant processed by pepsin, SG fermentation supernatant processed by trypsin, SG fermentation supernatant processed by pepsin and SG fermentation supernatant processed by pepsin; one sample solution per well and three duplicate wells per sample solution.

RPMI1640 complete medium containing only 20mg/mL of α -MM, PBS and the cell suspension of step (3) were used as blanks, respectively.

Placing in 5% CO₂And culturing in a cell culture box at 37 ℃ for 48 hours.

(5) Taking out 96-well cell plate, adding 15 μ L CCK8 (Guangzhou Yiyuan Biotechnology Co., Ltd.) into each well, mixing, adding 5% CO₂Culturing in a 37 ℃ cell culture box for 2h, taking out a 96-well cell plate, and detecting OD of each well by using an enzyme-labeling instrument (Bio-Reader3350)₄₅₀。

The results are shown in fig. 3 (as a blank control in the cell suspension of step (3)), and it can be seen that the lymphocytes obtained by stimulating porcine lymphoblasts with SG2B fermentation supernatant are significantly increased (P <0.05) compared with the control recombinant bacterium SG fermentation supernatant, whether treated with trypsin or pepsin; the fusion protein FBAPIL2 can obviously stimulate the porcine lymphocyte proliferation (P < 0.05).

In FIG. 3, untreated means fermentation supernatants that were not trypsinized and not pepsin treated.

Example 3 detection of the bacteriostatic Activity of the bovine antimicrobial peptide Interleukin 2 fusion protein (FBAPIL2)

Determination of bovine antimicrobial peptide Interleukin 2 fusion protein FBAPIL2 on Escherichia coli Standard (G)^-) (hereinafter abbreviated as S-G)^-) Escherichia coli resistant bacterium (G)^-) (hereinafter abbreviated as R-G)^-) Staphylococcus aureus (G)⁺) (hereinafter abbreviated as S-G)⁺) Staphylococcus aureus resistant bacterium (G)⁺) (hereinafter abbreviated as R-G)⁺) The specific method for inhibiting bacteria is as follows:

the 4 bacterial strains were first activated by inoculation and cultured in exponential growth phase (OD)₆₀₀About 0.5), and then diluted to OD with LB medium₆₀₀About 0.005, the diluted inoculum was inoculated into 96-well cell culture plates, 100. mu.L per well, one bacterium per 96-well cell culture plate.

The 96 well cell culture plates containing bacteria were treated as follows: adding 100 mu L of sample liquid into the experimental holes, and gently mixing uniformly, wherein each sample is provided with 3 repeated holes;

wherein the sample liquid was SG2B fermentation supernatant (i.e., untreated SG2B fermentation supernatant), trypsin-treated SG2B fermentation supernatant, pepsin-treated SG2B fermentation supernatant obtained in

steps

1 and 2 of example 2, and SG fermentation supernatant, trypsin-treated SG fermentation supernatant and pepsin-treated SG fermentation supernatant obtained in

steps

1 and 2 of example 2 were used as the respective empty controls.

Each strain was set with four antibiotic gradients as positive controls (antibiotics and LB broth); each strain was set with LB medium alone as a negative control and LB medium without any strain as a blank control.

Incubating 96-well cell culture plates in an incubator at 37 ℃ for 2h and 16h, and detecting OD of each well by using an enzyme labeling instrument (Bio-Reader3350)₆₀₀The value is obtained.

S-G^-And S-G⁺The four antibiotic gradients are shown in Table 1, R-G^-And R-G⁺The four antibiotic gradients are shown in table 2.

TABLE 1 antibiotic gradient for Standard strains

Note: in Table 1, "-" indicates that kanamycin is not contained.

TABLE 2 antibiotic gradient of drug resistant strains

The results showed that the OD of each sample solution of SG was equal to that of each of the four types of bacteria₆₀₀OD with negative control₆₀₀The difference between 2h and 16h shows that SG has no inhibition effect on the four bacteria; OD of each sample solution of SG2B₆₀₀OD at 2h and 16h were significantly lower than that of the empty control at the corresponding times₆₀₀(P<0.05)。

The results at 16h are shown in FIGS. 4-7 (the empty controls are SG fermentation supernatant), SG2B fermentation supernatant has inhibitory effect on all the four bacteria, and SG2B fermentation supernatant has better bacteriostatic effect when combined with antibiotics (ampicillin and kanamycin), which indicates that the fusion protein FBAPIL2 can inhibit Escherichia coli standard bacteria (G APIL2)^-) (hereinafter abbreviated as S-G)^-) Escherichia coli resistant bacterium (G)^-) (hereinafter abbreviated as R-G)^-) Staphylococcus aureus (G)⁺) (hereinafter abbreviated as S-G)⁺) Staphylococcus aureus resistant bacterium (G)⁺)。

Example 4 study of the biological Activity of bovine antimicrobial peptide Interleukin 2 fusion protein (FBAPIL2) in mice

1. Preparation of fermentation product

Fermentation: the recombinant bacterium SMDpG-2B (hereinafter referred to as SG2B) obtained in example 1 was activated and inoculated into a 100mL Erlenmeyer flask containing 30mLYPD medium, and cultured at 30 ℃ and 220rpm for 48 hours to OD₆₀₀About 25, SG2B fermentation broth was obtained.

And (3) fermenting the control recombinant bacterium SMDpG according to the method to obtain SG fermentation liquor.

2. Experimental ICR mice grouping

Taking 50 healthy female ICR mice of 18-20g and 3 weeks old, randomly grouping into 5 groups, wherein each group comprises 10 mice, the group number is 1-5, wherein group 1 and group 4 are SG negative control groups, group 3 is a vaccine negative control group, and group 2 and group 5 are experimental groups.

3. Mouse feeding and vaccination

According to grouping conditions, feeding fresh fermentation liquor into the stomach of a mouse by using a gavage needle, wherein each fermentation liquor is 0.6mL, marking the first gavage as the gavage day 0, and gavage once every two days for 4 weeks continuously (namely, the corresponding fermentation liquor is gavage respectively on the

gavage days

0, 3, 6, 9, 12, 15, 18, 21, 24 and 27, and the amount of each gavage is 0.6 mL/each). In order to ensure that the recombinant protein is not metabolized, fresh recombinant pichia pastoris liquid fermentation is carried out before the animal is gavaged every time.

When vaccination was performed, intramuscular injection (intramuscular injection) was performed only on day 7 of gavage, 0.2 mL/mouse.

The specific operation is as follows (Table 3), wherein the swine enzootic pneumonia (MP) vaccines are all products produced by Wapai Biotech group, Inc. (catalog number: 19200003).

TABLE 3 mouse gavage, vaccination dose

In table 3, the gavage is the inoculation mode of the fermentation broth, and 0.6 mL/piece is the inoculation dose of the fermentation broth; intramuscular injections were all vaccination regimens, 0.2 mL/single was vaccination dose of vaccine.

The tail venous blood of each mouse is collected before, on the 7 th, 14 th, 21 st and 28 th days of the intragastric perfusion respectively, and the following experimental contents are carried out: mixing 30 μ L of whole blood with 30 μ L of physiological saline, and making blood routine in a blood cell counter; flow cytometry is carried out on 50 mu L of whole blood; quantifying immune related genes in real time after extracting RNA from 100 mu L of whole blood; adding 100 μ L of whole blood into 1mLTRIZOL, mixing, and storing at-80 deg.C; plasma detection antibody was collected by centrifugation of 200. mu.L whole blood at low speed.

4. Toxicity attacking experiment

Killing Escherichia coli (Sichuan) with high drug resistanceUniversity animal epidemic disease prevention and control and food safety provided by Sichuan province key laboratory, catalog number: SCSU-ECOLI-HRL-1012) is inoculated into LB liquid culture medium containing 0.1mg/ml ampicillin and 0.1mg/ml kanamycin for activation, the activated fresh bacterial liquid is inoculated into LB liquid culture medium at 37 ℃, cultured at 1500rpm until logarithmic phase is centrifuged and the bacterial is collected, and is resuspended to 5.0 × 10 by using fresh LB liquid culture medium⁵CFU/ml, and obtaining the escherichia coli fermentation liquor. Preliminary experiments found that the half-lethal dose of E.coli fermentation broth to 3-week-old healthy female ICR mice was 0.1 ml. And (3) randomly selecting 5 mice from each group on the 30 th day of gastric lavage, injecting escherichia coli fermentation liquor into the abdominal cavity, wherein the injection amount of each mouse is half lethal dose, and the day of intraperitoneal injection is the 0 th day after toxin counteracting. The mice were observed every 24h for morbidity, and the survival rate of the mice was counted, and the organs inside the dead mice were dissected and observed for changes.

Inoculating high-drug-resistance lethal Staphylococcus aureus (provided by laboratory of animal epidemic disease prevention and control and food safety of Sichuan university, Sichuan province, and provided by Key laboratory of Sichuan province, catalog number: SCSU-STREPC-HRL-2026) into LB liquid culture medium containing 0.1mg/ml ampicillin and 0.1mg/ml kanamycin, activating, inoculating fresh bacterial liquid into LB liquid culture medium at 37 deg.C, culturing at 1500rpm to logarithmic phase, centrifuging, collecting thallus, and re-suspending to 5.0 × 10 with fresh LB liquid culture medium⁵CFU/ml to obtain the staphylococcus aureus fermentation liquor. Preliminary experiments have found that the half-lethal dose of Staphylococcus aureus broth to 3-week-old healthy female ICR mice is 0.2 ml. And (3) injecting staphylococcus aureus fermentation liquor into the abdominal cavity of the 5 mice which are not injected with the escherichia coli fermentation liquor and remain in each group on the 30 th day of gastric lavage, wherein the injection amount of each mouse is half lethal dose, and the day of intraperitoneal injection is also the 0 th day after toxicity attack. The mice were observed every 24h for morbidity, and the survival rate of the mice was counted, and the organs inside the dead mice were dissected and observed for changes.

5. Analysis of Experimental results

The result of conventional detection of peripheral blood leukocyte change of each group of mouse blood is shown in fig. 8, SG represents blank control bacteria fermentation broth, and the peripheral blood leukocyte content of experimental group of mice is significantly higher than that of SG negative control group and vaccine negative control group (P < 0.05); the SMDpG-2B fermentation product can effectively stimulate the proliferation of immune cells, and the fusion protein FBAPIL2 can effectively stimulate the proliferation of the immune cells.

Processing the sample by referring to the flow cytometry operation step, detecting the number of Th (CD4+ lymphocyte) cells and Tc (CD8+ lymphocyte) cells on a computer at low temperature in a dark place, and displaying the change of CD4+ and CD8+ lymphocytes in 10000 cells of randomly selected mouse peripheral blood as the flow cytometry result as shown in figures 9 and 10; as can be seen from the figure, the content of CD4+ and CD8+ in the peripheral blood of the immunized experimental mouse is significantly higher than that of the SG negative control group and the vaccine negative control group (P <0.05), and the peripheral blood reaches the peak value 21 or 28 days after immunization. The SMDpG-2B fermentation product has the function of stimulating the immune response of the experimental mice, and the fusion protein FBAPIL2 is shown to effectively stimulate the proliferation of immune cells.

The plasma collected by low-speed centrifugation is used for detecting nonspecific antibodies IgG, IgG1 and IgG2a and the antibody titer of a specific antibody MP according to the operation steps of an ELISA kit, wherein the kit is respectively a mouse immunoglobulin G1(IgG1) ELISA kit (cargo number 69-210245), a mouse immunoglobulin G (IgG) ELISA kit (cargo number 59-20037), a mouse immunoglobulin G (IgG2a) ELISA kit (cargo number 69-210250) and a swine mycoplasma antibody ELISA kit (cargo number 69-40349), and all the kits are products of Wuhan Michel biotechnology Limited.

The results are shown in fig. 11, 12 and 13, and show that the levels of IgG, IgG1 and IgG2a in peripheral serum of the immunized experimental mouse are significantly increased compared with those of the SG negative control group and the vaccine negative control group (P <0.05), and both reach a

peak value

14 or 28 days after immunization. The SMDpG-2B fermentation product can stimulate the immune mice to produce more IgG, IgG1 and IgG2a antibodies. Fig. 14 shows that the antibody titer of MP (mycoplasma) in peripheral serum of the immunized experimental mice gradually decreased with time, but the experimental group was significantly higher than the SG negative control group and the vaccine negative control group (P < 0.05). The SMDpG-2B fermentation product can obviously improve the immune response effect induced by the vaccine, and the fusion protein FBAPIL2 can effectively stimulate the proliferation of immune cells.

Detecting the expression condition of mouse immune related genes on the RNA level, extracting RNA from whole blood as a template, amplifying by using primers shown in table 4, and detecting the expression of the mouse immune related genes, wherein the reference gene is actin beta-actin.

Table 4 shows the primers

TNF- α is an important Th 1-type cytokine, and is mainly involved in the differentiation and cellular immunity of Th1 cells, the dynamic changes of the cytokine are shown in fig. 15, the experimental group is significantly higher than SG negative control group and vaccine negative control group (P <0.05), and the peak is reached on day 21 after immunization. The Th2 cells secrete IL4 cytokines, participate in the humoral immunity of the body, the dynamic changes of the Th2 cells are shown in fig. 16, the experimental group is significantly higher than the SG negative control group and the vaccine negative control group (P <0.05), and the peak value is reached in 14 to 21 days. The results show that the SMDpG-2B fermentation product can simultaneously promote cellular immunity and humoral immunity, namely the fusion protein FBAPIL2 can simultaneously promote cellular immunity and humoral immunity.

Fig. 17 shows the dynamic change of TLR gene expression level, and the results show that after immunization, the expression levels of TLR1 gene and TLR4 gene are significantly increased (P <0.05), and the TLR gene expression level of experimental mice is significantly increased (P <0.05) compared with SG negative control group and vaccine negative control group, and reaches the peak value 21 days after immunization.

Fig. 18 is a graph showing the dynamic change of the expression level of the immunological memory-associated genes, which generally shows that the levels of the IL-23 gene and IL-7 gene expression of the experimental group mice are significantly increased after the immunization compared with the SG negative control group and the vaccine negative control group (P < 0.05).

On the 5 th day after challenge, the survival rate of the mice in the experimental group is significantly higher than that of the SG negative control group and the vaccine negative control group (fig. 19) for the mice injected with the escherichia coli fermentation broth, and the survival rate of the mice in the experimental group is significantly higher than that of the SG negative control group and the vaccine negative control group for the mice injected with the staphylococcus aureus fermentation broth, which indicates that the SMDp2B in the experimental group can effectively protect the mice, so that the resistance of the mice to escherichia coli and staphylococcus aureus is significantly enhanced, namely the fusion protein FBAPIL2 can improve the survival rate of the mice after being attacked by pathogenic bacteria. The dead mice are dissected to find that the digestive tract in the abdominal cavity of the mice killed by the escherichia coli has obvious lesion, the spleen is blackened, the liver is blackened, the digestive tract in the abdominal cavity of the mice killed by the staphylococcus aureus infection has no obvious lesion, the spleen is blackened, and the liver is normal.

Example 5 study of the biological Activity of the fusion protein FBAPIL2 in piglets

1. Preparation of fermentation product

Fermentation: the recombinant bacterium SMDpG-2B (hereinafter referred to as SG2B) obtained in example 1 was activated and inoculated into a 100mL Erlenmeyer flask containing 30mLYPD medium, and cultured at 30 ℃ and 220rpm for 48 hours to OD₆₀₀About 40, SG2B fermentation liquor was obtained.

2. Fermentation liquor for feeding experimental animals

Selecting 17 healthy and Tibetan pigs with the weight of about 8kg and the age of 45 days, wherein the healthy and Tibetan pigs are provided by a simple yang base of a Sichuan province breeding pig performance measuring center and are randomly divided into an experimental group (9 pigs) and a control group (8 pigs). Feeding the fermentation liquor SG2B obtained in the step 1 to each pig in an experimental group, feeding the fermentation liquor SG2B obtained in the step 1 to each pig in a control group, wherein the feeding amount is 12.5ml/kg of body weight, feeding for 28 days, feeding once every 1 day, and recording the day before the first feeding as 0 day. 3-4mL of the forechamber venous blood were collected in vacuum tubes containing EDTA-K2 on

days

0, 7, 14, 28, and 42 for all piglets, respectively, after which the immune cells used for blood sampling were changed, and all piglets were weighed on

days

0, 28, and 56 for feeding.

3. Experimental piglet weight change

The experimental piglets were weighed at 0, 14, 28 and 42 days of feeding, and the results (fig. 20) showed that the piglets in the experimental group gained weight 1.43, 1.18 and 1.11 times of the piglets in the control group on 14, 28 and 42 days of feeding respectively, and the difference was significant (P < 0.05); SG2B shows that the piglet growth can be promoted more effectively.

4. Dynamic change of peripheral blood leukocyte number of experimental piglet

Blood samples taken at 0, 7, 14, 28 and 42 days of feeding were mixed together by group, and the number of leukocytes in the blood samples was measured with a conventional hematology analyzer. The results (fig. 21) show that the numbers of leukocytes in SG2B experimental group were significantly higher than those in SG control group (P <0.05) at day 42 of feeding. The fusion protein FBAPIL2 can effectively increase the number of peripheral blood immune cells of a subject, thereby enhancing immunity.

5. Detection of experimental piglet peripheral blood CD8+ T lymphocyte subset

Blood samples taken on

days

7, 14, 28 and 42 of feeding were mixed together in groups for flow cytometry of CD8+ T lymphocyte subpopulations in peripheral blood using Mouse Anti-Portene CD8a-SPRD (Southern Biotech, Cat. 4520-13) and 1. mu.l Mouse Anti-Portene CD3-FITC (Southern Biotech, Cat. 4510-02) and the specific steps were as follows:

(1) fresh anticoagulated piglet venous blood 100. mu.l (leukocyte count of about 10)⁵-10⁷One), 60. mu.l of physiological saline was added;

(2) pipetting 2. mu.l of Mouse Anti-Portone CD8a-SPRD (Southern Biotech, Cat. 4520-13) and 1. mu.l of Mouse Anti-Portone CD3-FITC (Southern Biotech, Cat. 4510-02) into 1.5ml EP tube, mixing well, and incubating for 20 min;

(3) adding 0.2ml of 10x erythrocyte lysate into a special test tube for a flow cytometer, adding 1.8ml of PBS, adding the incubated blood into the lysate, and performing lysis for 5min until the blood cells are completely lysed;

(4) centrifuge at 1500rpm for 5min, and discard the supernatant. Adding 2ml of PBS, blowing, beating and mixing uniformly, and suspending cells;

(5) centrifuging at 1500rpm for 5min, discarding supernatant, slightly blowing and mixing with 150 μ l PBS, and washing for 1-2 times, wherein the amount of washing solution is at least 5 times of cell precipitation volume;

(6) the cells were blown down with 150. mu.l PBS and mixed well for detection.

The results are shown in fig. 22, and the SG2B experimental group showed significantly higher numbers of CD8+ T lymphocytes than SG control group (P <0.05) on

days

7, 14, 28 and 42. The fusion protein FBAPIL2 can effectively stimulate the immune response function of piglets.

6. ELISA detection of CSF and PRRSV specific antibodies:

the plasma collected by low-speed centrifugation was subjected to detection of specific antibodies according to the procedure of an ELISA kit (swine fever antibody (CSF Ab) ELISA kit, porcine reproductive and respiratory syndrome virus ELISA kit).

Fig. 23 and 24 are graphs showing the dynamic changes of the antibodies specific to swine fever (CSF) and the antibodies specific to Porcine Reproductive and Respiratory Syndrome (PRRS) in the peripheral blood during the growth period of piglets, respectively, and it is evident that the amounts of both specific antibodies in the SG2B experimental group were significantly higher than those in the SG control group (P <0.05) at 0 days, 14 days, 28 days and 42 days of feeding. The fusion protein FBAPIL2 can remarkably enhance the immune response induced by the vaccine, thereby increasing the protective rate of the vaccine.

7. Variation of expression of immune-related genes

And (3) detecting the expression condition of the immune related genes of the piglets on the RNA level, wherein the immune related genes and primers are shown in the table 5, and the internal reference is actin beta-actin.

TABLE 5 quantitative primers

Fig. 25 shows the dynamic change of TLR gene expression level, and the results show that after immunization, the expression levels of TRL7 gene and TRL9 gene are significantly increased (P <0.05), and the expression level of TLR gene of experimental pig is significantly increased (P <0.05) compared with SG negative control group, and reaches

peak

14 or 28 days after immunization.

FIG. 26 shows the dynamic changes of the expression levels of the immunological memory-associated genes, and the results show that the expression levels of the CD45 gene and the CD62L gene of experimental pigs are obviously increased (P <0.05) after immunization and reach a peak value 14 days after immunization.

The change of the Bcl-2 gene of the immune signal transduction molecule is shown in figure 27, and the result shows that the expression level of the experimental pig is obviously increased compared with that of an SG negative control group after immunization (P < 0.05).

The fusion protein FBAPIL2 is shown to be capable of improving the expression of related genes after the immunization of the experimental swine fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS) vaccines.

Sequence listing

<110> Shenzhen, Shenzhou, Shenzhu

<120> preparation and application of fusion bovine antibacterial peptide and interleukin 2 co-expression recombinant yeast preparation

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Gln Leu Glu Pro Leu Leu Leu Asp Leu Gln Leu Leu Leu Lys Glu Val

35 40 45

Lys Asn Tyr Glu Asn Ala Asp Leu Ser Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Gln Ala Thr Glu Leu Lys His Leu Gln Cys Leu Val

65 70 75 80

Glu Glu Leu Lys Ala Leu Glu Gly Val Leu Asn Leu Gly Gln Ser Lys

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Asn Ser Asp Ser Ala Asn Ile Lys Glu Ser Met Asn Asn Ile Asn Val

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Thr Val Leu Glu Leu Lys Gly Ser Glu Thr Ser Phe Lys Cys Glu Tyr

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Asp Asp Glu Thr Val Thr Ala Val Glu Phe Leu Asn Lys Trp Ile Thr

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Phe Cys Gln Ser Ile Tyr Ser Thr Leu Thr Gly Ser Gly Glu Gly Arg

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Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

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Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala

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Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile

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Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp

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Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe

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Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser

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His

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ttacagttgc ttttgaagga agttaagaat tacgagaatg ctgatctctc caggatgctc 180

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Claims

1. A protein comprising bovine antimicrobial peptide and interleukin 2;

the protein is any one of the following a) or b):

a) the amino acid sequence consists of amino acid residues shown in a sequence 1 in a sequence table;

b) a) the N-terminal and/or C-terminal of the protein is connected with a label to obtain the fusion protein.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein: the coding sequence of the nucleic acid molecule is sequence 2 in the sequence table.

4. Any one of the following 1) -4):

1) an expression cassette comprising the nucleic acid molecule of claim 2 or 3;

2) a recombinant vector comprising the nucleic acid molecule of claim 2 or 3;

3) a recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 2 or 3;

4) the fermentation product of the recombinant bacteria in 3).

5. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 or 3 or the biomaterial of claim 4 for the preparation of a product for enhancing the immune competence of an animal;

the animal is a pig or a mouse.

6. Use according to claim 5, characterized in that: the animal immunity improving effect is at least one of the following M1-M5:

m1, inhibiting the growth of pathogenic microorganisms;

m2, promoting increase of immune cells;

m3, promoting vaccine-induced immune responses;

m4, promoting cellular and/or humoral immunity;

m5, increasing animal development and growth weight gain;

the pathogenic microorganism is Escherichia coli, Staphylococcus aureus, porcine reproductive and respiratory syndrome virus or classical swine fever virus;

the immune cell is lymphocyte, erythrocyte or leucocyte;

the antibody is in particular IgG, IgG1 and/or IgG2 a.

7. Any one of the following X1 or X2:

x1, a biological agent comprising the following X3a, X3b or X3 c:

x3a, the protein of claim 1;

x3b, the nucleic acid molecule of claim 2 or 3;

x3c, the biomaterial of claim 4;

x2, kit for improving animal immunity, consisting of the above X1 and antibiotics;

the animal is a pig or a mouse.