CN112195182A

CN112195182A - Preparation method and combined application of recombinant chicken interleukin 2 and interleukin 18 proteins

Info

Publication number: CN112195182A
Application number: CN202010813933.8A
Authority: CN
Inventors: 张艳雯; 郑一民; 周晴云; 覃静
Original assignee: Nanning University
Current assignee: Nanning University
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2021-01-08

Abstract

The present invention is in the field of biotechnology research, in particular to a method for preparing recombinant chicken interleukin 2 and interleukin 18 proteins and their combined application. The present invention provides a method for preparing recombinant chicken interleukin-2 and interleukin-18 proteins. The expression of recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 in Escherichia coli is improved by induction at low temperature, which greatly improves the protein content. It is an efficient preparation method for chicken interleukin-2 and interleukin-18 proteins. In addition, the present invention uses purified ChIL-2 and ChIL-18 proteins and eukaryotic plasmids expressing ChIL-2 and ChIL-18 proteins in combination with the Newcastle disease-infectious bronchitis dual live vaccine to immunize SPF chicks, so that the chickens can be decocted and enhanced. The IBV antibody titer can play an obvious immune-enhancing role in humoral immunity, and has a good application prospect.

Description

Preparation method and combined application of recombinant chicken interleukin 2 and interleukin 18 proteins

Technical Field

The invention relates to the field of biotechnology research, in particular to a preparation method and combined application of recombinant chicken interleukin 2 and interleukin 18 proteins.

Background

Chicken interleukin 2 and interleukin 18 are important cytokines, mainly produced by Th1 cells and some B cells. Interleukin 2 and interleukin 18 have wide biological activity, can promote the production of gamma-interferon (IFN-gamma), stimulate lymphocyte transformation, enhance the killing activity of NK cells, and have important effects in mediating cellular immunity and resisting microbial infection.

Interleukin-2 (IL-2) is the most clinically studied cytokine, a lymphokine secreted by T cells upon stimulation with antigen or mitogen. At present, IL-2 has good effects on the treatment of bacterial infection, virus infection and AIDS and the application as hepatitis B virus immunologic adjuvant.

Interleukin-18 (IL-18) is a substance with a molecular weight of about 18-19 kDa, which is isolated, purified and cloned by Okamura et al when mice livers are treated with heat-inactivated Propionibacterium acnes and lipopolysaccharides, and is named as gamma-interferon inducible factor (IGIF) because it is found to induce IFN-gamma production by T lymphocytes and NK cells. The study of chicken interleukin-18 (ChIL-18) began to be relatively late compared to mammals, and the study on the molecular aspect of chicken IL-18cDNA was not started until Schneider and the like clone to the chicken IL-18cDNA for the first time and successfully express in an Escherichia coli expression system in 2000. Domestic Liu Sheng Wang and the like are cloned to a chicken IL-18 mature protein gene with the length of about 510bp from chicken splenocytes, and guinea pigs are repeatedly injected after the expression and purification in escherichia coli to obtain an IL-18 polyclonal antibody, so that the domestic research on IL-18 enters the molecular level, and the research on the biological characteristics and the application of the IL-18 polyclonal antibody gradually becomes a hotspot.

Newcastle Disease (ND) is an acute, highly contagious avian infectious disease that has been seen to have four worldwide epidemics, causing significant economic losses to the poultry industry. The vaccine has the advantages that the vaccine is generated all the year round, and in recent years, the atypical newcastle disease caused by ND virulent strains is generated occasionally, the resistant chicken flocks are possibly infected for a long time and are frequently recurrent, so that the vaccine becomes an invisible infection source, the poultry breeding industry is seriously threatened, the method for preventing the newcastle disease through vaccine immunization is the most common method at present, but the immune effect is not ideal and even fails due to various internal and external factors, so the immune effect of the newcastle disease vaccine is improved, and the prevention of the newcastle disease is always concerned. Infectious Bronchitis (IB) is an acute and highly infectious respiratory disease of chickens, is distributed worldwide, and has great harm to the chicken industry. This disease often causes severe respiratory distress to chicks and can lead to decreased egg production by the laying hens. At present, the main disease forms of IB in China are dyspnea and kidney swelling, namely respiratory type and kidney type. The NIB has gradually spread around the world since its discovery in the United states in 1950, and has become the most prevalent type of IB in recent years.

Therefore, the search for a method for efficiently preparing chicken interleukin 2 and interleukin 18 protein and the search for the application direction of chicken interleukin 2 and interleukin 18 are the technical problems which need to be solved urgently at present.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a preparation method and combined application of recombinant chicken interleukin 2 and interleukin 18 proteins, which are realized by the following technical scheme:

a preparation method of recombinant chicken interleukin 2 and interleukin 18 protein comprises the following steps:

(1) separating and culturing SPF chicken spleen lymphocytes;

(2) extracting total RNA of cells;

(3) amplifying ChIL-2 and ChIL-18 genes by RT-PCR;

(4) cloning and enzyme digestion identification of a PCR product;

(5) sequencing analysis of positive clones of ChIL-2 and ChIL-18 genes;

(6) constructing recombinant expression plasmids pET32a-ChIL-2 and pET28 a-ChIL-18;

(7) the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 were expressed in E.coli.

Further, the separation and culture of the SPF chicken spleen lymphocytes in the step (1) are specifically to take blood from the heart of the chicken and kill the heart, aseptically take the chicken spleen and place the chicken spleen in cold Hanks liquid, after separating the envelope and the adipose tissue, grind the chicken spleen on a 200-mesh copper mesh to prepare single cell suspension, centrifuge the chicken spleen at 2000rpm/min for 15min, suspend the precipitated cells in RPMI1640 culture solution containing 10% fetal calf serum, take a centrifuge tube, add 4ml of lymphocyte separation solution in advance, slowly add the equal volume of cell suspension to the lymphocyte separation solution, perform density gradient centrifugation, centrifuge the chicken spleen cell suspension at 2000rpm/min for 15min, collect lymphocytes, centrifuge and wash the cells for 2 times by using the RPMI1640 culture solution, count the number of living cells, adjust the cell concentration to 1 × 107 cells/ml, add 2ml of 6-well cell culture plates to each well, place the cells in a 40 ℃, 5% CO2 cell culture box, and culture.

Further, the RT-PCR of the step (2) amplifies ChIL-2 and ChIL-18 genes, specifically, collecting cultured cells at 6h and 20h respectively, centrifuging at 4 ℃ and 2000rpm/min for 15min, resuspending the cells with 1ml of RPMI1640, and extracting total RNA according to Trizol Reagent instructions: adding 1ml of Trizol into 1 × 107 cells, oscillating vigorously, centrifuging at 12000rpm/min at 4 ℃ for 10min, sucking the supernatant to a new Ep tube, adding 200 μ l of chloroform, oscillating vigorously, standing at 15-30 ℃ for 2-3 min, centrifuging at 12000rpm/min at 4 ℃ for 15min, carefully sucking the uppermost colorless aqueous phase layer to the new tube, adding 1-fold volume of isopropanol, mixing uniformly, centrifuging at 12000rpm/min at 4 ℃ for 10min, discarding the supernatant, adding 75% of ethanol to wash the precipitate of the RNA, pouring off the ethanol, drying in the air or drying in vacuum for 10-15 min, adding 25u of 1 DEPC-treated water-soluble RNA, checking the RNA extraction effect by electrophoresis, and storing the extracted total RNA of chicken spleen lymphocytes at-70 ℃ for later use.

Further, the ChIL-2 and ChIL-18 genes are amplified by the RT-PCR in the step (3), and the method specifically comprises the steps of firstly, designing and synthesizing a primer, and secondly, amplifying the genes by the RT-PCR;

the Primer design and synthesis specifically comprises the following steps of designing and amplifying specific primers of ChIL-2 and ChIL-18 by using Primer software according to gene sequences of ChIL-2 and ChIL-18 in GeneBank, synthesizing the primers by Shanghai biological engineering Limited company, and specifically comprising the following primers for amplifying the genes of ChIL-2 and ChIL-18:

dissolving dried RNA with 15-20 mul DEPC water, sucking 11.5 mul RNA, adding the RNA into a new PCR tube, adding 2 mul primers, taking out a mixture at 70 ℃ for 5min in a PCR instrument, sequentially adding 2 mul dNTP, 1 mul reverse transcriptase, 0.5 mul RNase inhibitor and 4 mul 5 xbuffer after ice bath for 1min, putting a sample into the PCR instrument, preserving at 37 ℃ for 1h and 95 ℃ for 10min, and preserving at 4 ℃ or-20 ℃ for later use; performing Taq enzyme PCR amplification on the obtained reverse transcription cDNA product;

the PCR reaction system is shown below: 50 μ L

The PCR reaction conditions were as follows:

after the reaction, the PCR amplification product was identified by 1% agarose gel electrophoresis, and the DNA fragment was recovered.

Further, the cloning and enzyme digestion identification of the PCR product in the step (4) specifically includes cloning the recovered DNA fragment into the pMD19-T vector, and specifically includes the following operations:

the PCR product plus A system is as follows: 10 μ L

Ligation of pMD19-T vector: 10 μ L

The reaction was carried out at 16 ℃ for 30min or at 4 ℃ overnight to transform DH10B competent cells.

Further, according to the positive clone sequencing analysis of the ChIL-2 and ChIL-18 genes in the step (5), after the plate after transformation is cultured for 16 hours, a single colony is selected to be placed in 10 mu L of sterile water, 1 mu L of bacterial liquid is taken as a template, colony PCR identification is carried out on a Taq DNA polymerase system by using a specific primer, the positive bacteria are taken to be transferred to a fresh culture medium, when the bacterial liquid is cultured to a proper concentration, small-scale extraction of plasmids is carried out, a proper enzyme cutting site is selected for enzyme cutting identification, and PCR fragments are sequenced.

Further, the recombinant expression plasmids pET32a-ChIL-2 and pET28a-ChIL-18 in the step (6) are constructed, specifically, pMD19-T-ChIL-2 and pMD19-T-ChIL-18 are used as templates, PCR amplification is carried out by using the following primers, target gene fragments are recovered from agarose gel, BamHI and Sall are used for carrying out double enzyme digestion on pET32a and pET28a, the target gene fragments ChIL-2, a carrier pET32a, ChIL-18 and a carrier pET28a are respectively prepared into a ligation reaction system, the ligation reaction system is connected at 4 ℃ for one night, ligation reaction products are transferred into a competent cell 10B, a plate is coated, the next day is carried out overnight, a single colony is picked for colony PCR identification, a DH colony is taken, a fresh culture medium is inoculated to a DH colony, and when the DH bacterial liquid is cultured to a proper concentration, a small amount of the plasmid is extracted. The restriction sites were selected for restriction identification and sequencing, and the primers used to construct pET32a-ChIL-2 and pET28a-ChIL-18 were as follows:

further, the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 of the step (7) are expressed in Escherichia coli, specifically, the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 are transformed into Escherichia coli BL21, plates are coated, the temperature is 37 ℃ overnight, a single colony is picked into an LB culture medium the next day, when the bacterial liquid concentration A600 is 1.5, IPTG is added to the final concentration of 0.1mmol/L, induction culture is carried out at 20 ℃ overnight, and proteins are collected.

Further, the method comprises the step (8) of purifying recombinant ChIL-2 and ChIL-18 proteins, specifically, centrifuging 10,000g for 10min to collect thalli, re-suspending the collected thalli by 50ml of ultrasonication buffer solution, carrying out ultrasonication in an ice bath, centrifuging 14,000g of lysate for 20min to remove cell debris, filtering supernate after centrifugation by a 0.22um filter membrane, and purifying by referring to a His Tag fusion protein purification operation manual.

The chicken interleukin 2 and chicken interleukin 18 protein are applied to the aspect of combined immunization of SPF chicks with newcastle disease-infectious bronchitis bigeminy live vaccines, and particularly, the SPF chicks are fed by purified ChIL-2 and ChIL-18 protein and eukaryotic plasmids expressing the ChIL-2 and ChIL-18 protein, and are combined with the newcastle disease-infectious bronchitis bigeminy live vaccines to immunize the SPF chicks, after the combined use, the IBV antibody titer of the chickens can be obviously enhanced, and the obvious immune enhancement effect can be achieved in the aspect of humoral immunity. In addition, the purified ChIL-2 and ChIL-18 proteins and eukaryotic plasmids expressing the ChIL-2 and ChIL-18 proteins in the application can achieve the immune enhancement effect by feeding SPF chicks, so that the problem that the conventional immune adjuvant needs to be injected is avoided, the administration is more convenient, and the damage to the chicks is reduced.

Compared with the prior art, the invention has the technical effects that:

the invention provides a preparation method of recombinant chicken interleukin 2 and interleukin 18 protein, which comprises the following steps: (1) separating and culturing SPF chicken spleen lymphocytes; (2) extracting total RNA of cells; (3) amplifying ChIL-2 and ChIL-18 genes by RT-PCR; (4) cloning and enzyme digestion identification of a PCR product; (5) sequencing analysis of positive clones of ChIL-2 and ChIL-18 genes; (6) constructing recombinant expression plasmids pET32a-ChIL-2 and pET28 a-ChIL-18; (7) the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 were expressed in E.coli. The recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 are promoted to be expressed in escherichia coli through low-temperature induction, the production amount of protein is greatly promoted, and the method is an efficient preparation method of chicken interleukin 2 and interleukin 18 protein. In addition, the purified ChIL-2 and ChIL-18 proteins, eukaryotic plasmids expressing the ChIL-2 and ChIL-18 proteins and the Newcastle disease-infectious bronchitis bigeminal live vaccine are used for immunizing the SPF chicks in a combined manner, the IBV antibody titer of the chickens can be obviously enhanced after the combination, and the obvious immune enhancement effect can be achieved in the aspect of humoral immunity. In addition, the purified ChIL-2 and ChIL-18 proteins and eukaryotic plasmids expressing the ChIL-2 and ChIL-18 proteins in the application can achieve the immune enhancement effect by feeding SPF chicks, so that the problem that the conventional immune adjuvant needs to be injected is avoided, the administration is more convenient, and the damage to the chicks is reduced.

Drawings

FIG. 1 is an agarose gel electrophoresis of RT-PCR products;

FIG. 2 is the electrophoretic analysis chart of recombinant plasmid digestion pMD19-T-ChIL-2 and pMD 19-T-ChIL-18;

FIG. 3 is the restriction enzyme electrophoresis analysis chart of recombinant plasmids pET32a-ChIL-2 and pET28 a-ChIL-18;

FIG. 4 is a diagram of induced expression and purification of recombinant proteins;

FIG. 5 is a western blot detection diagram;

FIG. 6 is a diagram of the restriction enzyme electrophoresis analysis of the recombinant plasmids pVAX1-ChIL-2 and pVAX 1-ChIL-18;

FIG. 7 is a graph of protein expression detected by indirect immunofluorescence after transfection of Vero cells;

FIG. 8 is a graph of the levels of NDV HI antibodies in the serum of various groups of chickens at different times;

FIG. 9 is a graph of serum IBV IgG antibody levels.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.

Examples

1 materials of the experiment

1.1 Experimental animals, viruses and vaccines

14-day-old SPF chicks were purchased from Kyunjiang Bomeilai bioproduct, Inc.; the combined live vaccine (LaSota + H120 strain) for Newcastle disease and infectious bronchitis of chicken is purchased from Jiujiang Bomeilai biological products, Co., Ltd; NDV F48E9 strain, supplied by the animal husbandry and veterinary institute of agricultural academy of sciences, north hui; the IBDV standard virulent strain BC6/85 is purchased from Chinese veterinary drug inspection institute; the IBDV polyprotein DNA vaccine is prepared by extracting IBDV polyprotein gene eukaryotic expression plasmid pCI-VP2/4/3 by alkaline lysis method, purifying, measuring its concentration by ultraviolet spectrophotometer, and diluting properly.

1.2 bacterial species and cloning vectors

Escherichia coli BL21, Escherichia coli DH10B, pET32a prokaryotic expression vector, pET28a prokaryotic expression vector, pVAX1 eukaryotic expression vector are preserved for the university of Wuhan university institute of Life sciences Panz book professor laboratory; pMD19-T vector was purchased from Takara, a bioengineering great Lianbao.

1.3 Main instruments

A small-sized desk centrifuge, a low-temperature high-speed centrifuge, a liquid-transferring gun, a balance, a pH meter, an electric heating constant-temperature incubator, a magnetic stirrer, an autoclave, a decoloring shaking table, a multipurpose rotary shaking table, an ice maker, a nucleic acid electrophoresis apparatus, an electrophoresis tank, a protein electrophoresis apparatus, a PCR apparatus, a water bath and an ultrasonic cell crusher; a gel imaging system.

1.4 tool enzymes and other reagents

Omega plasmid miniextraction kit, Omega plasmid macroextraction kit, DNA gel recovery kit, conventional restriction enzymes (Kpn I, SalI, BamHI, EcoRI, Pst I, Mlu I, Xba I), DNA polymerase for PCR (Taq enzyme), protein ladder, dATP (R0141), T4 DNA ligase, DNA ladder, dNTPs, yeast extract, NaCl, tryptone, phosphate buffer, glycine, Tris base, methanol, canavalin A, tetramethyl azoazolate.

2 method

2.1 cloning of the ChIL-2 and ChIL-18 genes

2.1.1 isolation and culture of SPF Chicken spleen lymphocytes

Collecting blood from heart of chicken, killing, aseptically placing spleen of chicken in cold Hanks solution, separating envelope and adipose tissue, grinding on 200 mesh copper net to obtain single cell suspension, centrifuging at 2000rpm/min for 15min, and suspending the precipitated cell in suspensionAdding 4ml of lymphocyte separation liquid into RPMI1640 culture liquid containing 10% fetal calf serum, taking a centrifuge tube, slowly adding equal volume of cell suspension onto the lymphocyte separation liquid, performing density gradient centrifugation, centrifuging at 2000rpm/min for 15min, collecting lymphocytes, centrifuging and washing with RPMI1640 culture liquid for 2 times, counting the number of living cells, adjusting the cell concentration to 1 × 10⁷Adding the cells per ml to a 6-well cell culture plate, wherein each well contains 2ml of the cells, placing the cells at 40 ℃ and 5% CO₂Culturing in a cell culture box.

2.1.2 extraction of Total RNA from cells

The cultured cells were harvested at 6h, 20h, centrifuged at 2000rpm/min at 4 ℃ for 15min, resuspended in 1ml RPMI1640, and total RNA extracted according to Trizol Reagent instructions: 1X 10⁷Adding 1ml of Trizol into each cell, oscillating vigorously, centrifuging at 12000rpm/min at 4 ℃ for 10min, sucking the supernatant into a new Ep tube, adding 200 mu l of chloroform, oscillating vigorously, and standing at 15-30 ℃ for 2-3 min. Centrifuging at 12000rpm/min at 4 ℃ for 15min, carefully absorbing the uppermost colorless aqueous phase layer to a new tube, adding 1-time volume of isopropanol, uniformly mixing, centrifuging at 12000rpm/min at 4 ℃ for 10min, discarding supernatant, adding 75% ethanol to wash the precipitate of RNA, pouring off the ethanol, drying in air or drying in vacuum for 10-15 min, adding 25u1 DEPC-treated water to dissolve the RNA, detecting the RNA extraction effect by agarose electrophoresis, and storing the extracted total RNA of the chicken spleen lymphocytes at-70 ℃ for later use.

2.1.3 RT-PCR amplification of ChIL-2 and ChIL-18 genes

Primer design and synthesis

According to gene sequences of ChIL-2 and ChIL-18 in GeneBank, Primer software is used for designing and amplifying specific primers of the ChIL-2 and ChIL-18, the detailed description is shown in Table 1, and the primers are synthesized by Shanghai biological engineering Limited.

TABLE 1 primers for amplification of ChIL-2 and ChIL-18 genes

② RT-PCR amplified gene

The dried RNA was dissolved in 15-20. mu.l DEPC water and 11.5. mu.l of RNA was pipetted into a new PCR tube and 2. mu.l of primers were added to the tube and the tube was incubated at 70 ℃ for 5min in a PCR apparatus. The mixture was taken out, ice-washed for 1min, then 2. mu.l dNTP, 1. mu.l reverse transcriptase, 0.5. mu.l RNase inhibitor and 4. mu.l 5 XBuffer were added in order, the sample was placed in a PCR instrument, 1h at 37 ℃ and 10min at 95 ℃ and stored at 4 ℃ or-20 ℃ for further use.

The obtained reverse transcription cDNA product is amplified by Taq enzyme PCR.

The PCR reaction system is shown below: 50 μ L

The PCR reaction conditions were as follows:

2.1.4 cloning and restriction enzyme identification of PCR products

Cloning the recovered DNA fragment into a pMD19-T vector, and specifically operating as follows:

the PCR product plus A system is as follows: 10 μ L

Ligation of pMD19-T vector: 10 μ L

2.1.5 sequencing analysis of positive clones of ChIL-2 and ChIL-18 genes

After the transformed plate is cultured for 16 hours, a single colony is selected to be placed in 10 mu L of sterile water, 1 mu L of bacterial liquid is taken as a template, and colony PCR identification is carried out by using a specific primer in a Taq DNA polymerase system. The positive bacteria were transferred to fresh medium and when the bacteria were cultured to the appropriate concentration, a small amount of plasmid was extracted (refer to the Omega kit instructions). Selecting proper enzyme cutting sites for enzyme cutting identification, and sequencing the PCR fragments.

2.2 expression, purification and biological Activity detection of ChIL-2 and ChIL-18 proteins

2.2.1 construction of recombinant expression plasmids pET32a-ChIL-2 and pET28a-ChIL-18

PCR amplification is carried out by taking pMD19-T-ChIL-2 and pMD19-T-ChIL-18 as templates and using a primer of table 2, and a target gene fragment is recovered by agarose gel. Both pET32a and pET28a were digested with BamHI and Sall.

TABLE 2 primers used for the construction of pET32a-ChIL-2 and pET28a-ChIL-18

The target gene fragment ChIL-2 and the vector pET32a, and the ChIL-18 and the vector pET28a are respectively prepared into a connection reaction system, and are connected at 4 ℃ overnight. Ligation products were transferred to competent cell DH10B, plated and incubated overnight at 37 ℃. And (3) selecting a single colony for colony PCR identification on the next day, inoculating a fresh culture medium to the positive colony, and performing small-amount plasmid extraction when the bacterial liquid is cultured to a proper concentration. Selecting enzyme cutting sites for enzyme cutting identification and sequencing.

2.2.2.2 expression of recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 in E.coli

The recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 were transformed into E.coli BL21, plated and incubated overnight at 37 ℃. Picking a single colony to an LB culture medium on the next day until the concentration of the bacterial liquid is A₆₀₀When the concentration was 1.5, IPTG was added to a final concentration of 0.1mmol/L, and the culture was induced overnight at 20 ℃. And taking part of the bacterial liquid to perform SDS-PAGE analysis and Western blot identification.

2.2.3 recombinant ChIL-2 and ChIL-18 protein purification

The cells were collected by centrifugation at 10,000g for 10 min. The collected cells were resuspended in 50ml of sonication buffer, sonicated in an ice bath, the lysate was centrifuged at 14,000g for 20min to remove cell debris, and the supernatant after centrifugation was filtered through a 0.22um filter membrane and purified according to the manual of His. Tag fusion protein purification.

② the protein concentration determination by Brandford method of recombinant protein expression

Sucking 20mg/ml standard bovine serum albumin 6. mu.l, diluting 40 times to 0.5mg/ml with 0.15mmol/L NaCl, adding 10. mu.l, 20. mu.l, 30. mu.l, 40. mu.l into two groups of 4 test tubes respectively, then supplementing to total volume 200. mu.l with 0.15mmol/L NaCl, and taking one test tube and adding only 200. mu.l of 0.15mmol/L NaCl for zero adjustment; taking 20 μ l of the purified product, and complementing the purified product to a total volume of 200 μ l by 0.15 mmol/LNaCl; adding 2ml of Coomassie brilliant blue G-250 dye solution into each tube, shaking, mixing uniformly, and standing at room temperature for 30 min; in that

Reading the A590 value in a series600 full-wavelength spectrophotometer, and calculating the protein content of the sample.

2.2.4 identification of expression products of the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18

(ii) SDS-PAGE analysis

Filling the protein vertical electrophoresis tank according to the specification, closing the peripheral gap, pouring 13% of separation gel, preparing 5% of lamination gel after the separation gel is completely polymerized, uniformly mixing, pouring into a glass interlayer, and immediately inserting into a comb. And (3) after the laminated gel is polymerized, pulling out a comb, sequentially spotting and carrying out electrophoresis, taking out the gel after the electrophoresis is finished, gently shaking and dyeing the gel by using Coomassie brilliant blue R250 dye solution, and observing a protein band after the gel is fully decolored by using decolorant.

②Western blot

Preparing gel by SDS-PAGE as before; preparing Whatman 3mm filter paper and a PVDF membrane with proper sizes, taking down the gel after electrophoresis, transferring the gel, the filter paper, a spongy cushion and the PVDF membrane pretreated by methanol into a membrane transferring buffer solution of an ice bath, and soaking for 15-20 min; assembling a device for transferring a film from a negative electrode to a positive electrode in the order of "foam pad → 3 layer of filter paper → gel → PVDF film → 3 layer of filter paper → foam pad", while taking care to avoid the generation of air bubbles; rotating the membrane with 80V constant pressure ice bath for 60-120min (adjusting time according to the size of the target protein); and (3) sealing: taking out the PVDF membrane, sealing with 5% (w/v) skimmed milk, and standing at 4 deg.C overnight or 37 deg.C for 120 min; primary anti-reaction: washing the sealed PVDF membrane with PBST for 10min × 1 times, adding diluted primary antibody, incubating for 90min at 37 ℃, and washing with PBST for 10min × 4 times; secondary antibody reaction: adding diluted secondary antibody labeled by HRP, incubating for 60min at 37 ℃, and washing for 10min multiplied by 5 times by PBST; ECL chemiluminescence detection.

2.2.5 detection of biological Activity of recombinant ChIL-2 and ChIL-18 proteins on splenic lymphocytes of chickens

The activity was measured by lymphocyte proliferation assay, and lymphocyte proliferation was measured by MTT method. Collecting 45-60 days old SPF chicken, slaughtering, placing spleen into a sterilized small beaker, cutting with scissors, gently filtering with four layers of sterile gauze, and collecting filtrate. Centrifuging the filtrate at 2500rpm for 5-10 min. The supernatant was discarded. Then, an amount of RPMI1640 medium was added for resuspension. Adding about 4-5ml of lymphocyte separation solution into a new 15ml centrifuge tube, slowly adding equal volume of lymphocyte suspension along the wall to cover the upper layer of the separation solution, then centrifuging at 2000rpm for 15min, and slightly sucking the middle gray fog layer and adding into another sterile centrifuge tube. 2000rpm, centrifuge for 5min and discard the supernatant. An amount of PBS solution was added, the cells were resuspended and washed and centrifuged at 2000rpm for 10min and the supernatant removed. After 5ml of a medium containing 5. mu.g/ml of concanavalin (ConA) and 10% fetal bovine serum-containing RPMI1640 was added, the mixture was transferred to a cell culture dish, and cells were counted on a cell counting plate to adjust the cell density to 2X 106 cells, which were used as responder cells. After the preparation, the 96-well plate was incubated at 37 ℃ in a 5% CO2 incubator for 48 hours. Then, 10 to 15. mu.l of MTT solution with a concentration of 5mg/ml was added to each well, and the incubation was continued for 3 hours. Adding 100 μ l of lysate, placing in incubator, reacting for 2h at constant temperature, detecting the light absorption value (A570nm) at 570nm with ELISA, and calculating the Stimulation Index (SI) according to the following formula: SI ═ (mean OD value of experimental group-mean OD value of blank wells)/(mean OD value of negative control group-mean OD value of blank wells).

2.3 eukaryotic expression of ChIL-2 and ChIL-18 genes

2.3.1 construction of pVAX1-ChIL-2 and pVAX1-ChIL-18 plasmids

pMD19-T-ChIL-2 and pMD19-T-ChIL-18 are used as templates, PCR amplification is carried out by using primers in a table 3, a target gene fragment recovered by agarose gel is connected with a pVAX1 vector after enzyme digestion, transformed into competent cells, coated on a flat plate and cultured overnight at 37 ℃.

TABLE 3 primers used to construct pVAX1-ChIL-2 and pVAX1-ChIL-18

2.3.2 identification of pVAX1-ChIL-2 and pVAX1-ChIL-18 plasmids

The reference Omega plasmid miniprep kit requires a small amount of plasmid to be extracted for enzyme digestion identification and sent for sequencing.

2.3.3 transfection of recombinant plasmids into Vero cells

One day before transfection, Vero cells were trypsinized and counted, inoculated onto 6-well plates in a medium of 1640 medium without double antibody and containing 10% calf serum in 5% CO₂Culturing in an incubator for 24h until about 90% of the cells are confluent; for each well of cells, 4.0. mu.g of DNA (pcDNA3.1(+)) was diluted with 250. mu.l of serum-free 1640 medium; lipofectamine2000 transfection reagent was gently mixed and the cells were diluted with 250. mu.l serum-free 1640 medium per well. Mixing, and incubating at room temperature for 5 min; mixing the diluted DNA and the diluted Lipofectamine2000 transfection reagent, and standing at room temperature for 20 min; sucking out the old culture solution in the plate, washing twice by using a serum-free 1640 culture medium, and adding 2ml of the serum-free 1640 culture medium; add 500 μm of liposome/DNA mixture to each well and mix well with gentle shaking. Placing in 5% CO₂And incubating in an incubator for 24 h.

2.3.4 Mass extraction of recombinant plasmids pVAX1-ChIL-2 and pVAX1-ChIL-18

The operation is carried out according to the instruction of the Endo-Free Plasmid DNA Maxi Kit Plasmid large-volume Kit.

2.4 experiments on chicken immunization and challenge infection

2.4.1 grouping, immunization and serum Collection of Experimental Chicken of Newcastle disease

14 day old SPF chickens 84 were randomly divided into 7 groups of 12 chickens each. The experimental groups are shown in Table 4.

TABLE 4 grouping and administration of experimental animals

Seven groups of chickens were all administered at 14 days of age according to the above table, wherein the vaccine was instilled to the eyes, other samples were taken orally, and non-anticoagulant isolated sera were taken from the infrapterygeal vein or heart 1 day before and 4, 7, 14, 21, 28 and 42 days after inoculation, and the hemagglutination titer of NDV, ELISA method was used to determine the IBV antibody level in the sera.

2.4.2 serum NDV antibody detection

As determined by hemagglutination assay. Mu.l PBS was added to 1-11 wells of the microplate and 50. mu.l PBS was added to 12 th well. Sucking 25 mul serum into the 1 st hole, repeatedly blowing and beating for 6 times, fully mixing, sucking 25 mul serum into the 2 nd hole, sequentially diluting to the 10 th hole in a double way, sucking 25 mul serum from the 10 th hole and discarding. Adding 25 μ l of diluent containing 4 units of antigen into 1-11 wells, shaking on a micro-oscillator for 2min, and standing at room temperature for 30 min. Adding 25 μ l of chicken red blood cell suspension with volume fraction of 1% into each well, mixing, standing at room temperature for about 40min, and allowing the control red blood cells to appear to sink to the bottom of the well in a button shape. And (4) judging a result: the micro reaction plate is inclined at 45 degrees, and the red blood cells which are settled at the bottom of the hole flow downwards along the inclined surface in a linear way to form precipitates, which indicates that the red blood cells are not or not completely agglutinated by the viruses; the red blood cells spread on the bottom of the well, and are condensed into a uniform thin layer, and the red blood cells do not flow after being inclined, which indicates that the red blood cells are condensed by the virus. The highest dilution of serum capable of completely inhibiting the action of 4 units of virus agglutinated erythrocytes was taken as the erythrocyte agglutination inhibition titer of the serum, and the highest dilution of serum capable of completely inhibiting 4 units of antigen was taken as the HI titer as the logarithm to the base 2 (Log 2). The serum titer of the negative control hole is not more than 21og2, the serum error of the positive control hole is not more than 1 titer, and the test result is effective. A HI value of less than or equal to 21og2 determines that the HI test is negative; HI equal to 31og2 is suspicious and the test is repeated; a HI value greater than or equal to 41og2 is positive.

2.4.3 detection of Newcastle disease vaccine serum IBV antibody

And (3) taking the enzyme-labeled lath with the required dosage, setting a blank control 1 hole and 2 negative/positive controls respectively, sealing the unused lath as soon as possible, and storing at 2-8 ℃. Adding 100 mul of sample diluent into the blank control hole; adding 100 μ l of negative and positive control into the negative and positive control wells respectively; the diluted sample was added in 100. mu.l per well. Mixing, and reacting at 37 deg.C for 30 min. And (4) deducting liquid in the holes, filling washing liquid in each hole, standing for 30 seconds, then discarding, repeatedly washing for 5 times, and patting dry. Enzyme label was added in 100. mu.l per well (except for blank wells). The reaction mixture was left at 37 ℃ for 30 min. And (5) washing, which is the same as the step. Adding 50 μ l of substrate solution A and 50 μ l of substrate solution B into each well in sequence, mixing, and reacting at 37 deg.C in dark for 10 min. Add 50. mu.l of stop solution to each well, mix well, zero with blank well, and measure absorbance (A value) at 450nm (630 nm can be used as reference wavelength).

2.4.4 NDV challenge infection test

On the 48 th day after immunization, the virulent strain F48E9 of Newcastle disease was diluted to 10 degrees with sterilized normal saline⁶LD50/ml, each group of chickens was challenged by eye-drop nasal route, each 0.1ml, and then the mental state, diet and death status of the flock were observed and recorded, and after death, they were examined and pathological changes were recorded. And killing the surviving chickens in the seventh day after the virus attack, performing autopsy and recording pathological changes.

2.4.5 grouping and challenge infection test of infectious bursal disease experimental chickens

14 day old SPF chickens 84 were randomly divided into 7 groups of 12 chickens each. The experimental groups are shown in Table 5.

TABLE 5 grouping and administration of Experimental animals

All immunization groups were first immunized by oral IBDV polyprotein DNA vaccine at 14 days of age and second immunized by oral administration after 14 days. On the 17 th day after the second immunization, the IBDV standard virulent strain BC6/85 is used for attacking, after the attack, the mental state, the diet state and the death condition of the chicken flocks are observed and recorded, the surviving chickens are killed on the 6 th day, and the immune protection rate is calculated.

3 results of the experiment

3.1 cloning of the ChIL-2 and ChIL-18 genes

3.1.1 PCR identification of ChIL-2 and ChIL-18 genes

The RT-PCR product is checked by agarose gel electrophoresis, and target gene bands can be seen at 429bp and 593 bp. See figure 1 for details.

3.1.2 sequencing identification of ChIL-2 and ChIL-18 genes

Sequencing results show that the full length of the ChIL-2 gene fragment cloned in the experiment is 429bp, and the full length of the ChIL-18 gene fragment is 593 bp. The sequencing results were as follows:

ChIL-2：

ATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAATGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGAAAAATGGAAAACTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAATAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAChIL-18：

ATGAGCTGTGAAGAGATCGCTGTGTGTGCAGTACGGCTTAGAGAAAACCTCTGCCTCTATTTTGAAGATGATGAGCTGGAATGCGATGCCTTTTGTAAGGATAAAACTATCAAACGATTCTTTCGAAACGTCAATAGCCAGTTGCTTGTGGTTCGTCCAGATTTAAACGTGGCAGCTTTTGAAGATGTAACAGATCAGGAGGTGAAATCTGGCAGTGGAATGTACTTCGACATTCACTGTTACAAAACCACCGCGCCTTCAGCAGGGATGCCTGTTGCATTCAGCGTCCAGGTAGAAGATAAGAGTTACTACATGTGTTGTGAGAAAGAGCATGGGAAAATGGTTGTTCGATTTAGGGAAGGAGAAGTTCCCAAAGACATTCCTGGTGAAAGTAACATCATATTTTTCAAAAAGACATTTACATCTTGCAGCTCCAAGGCTTTTAAGTTCGAGTACTCACTTGAACAAGGAATGTTCTTGGCCTTTGAGGAAGAAGACTCCTTAAGAAAACTAATTTTAAAGAAACTGCCGAGAGAAGATGAAGTTGATGAAACCACAAAATTCGTAACAAGTCATAATGAAAGGCACAACCTA

3.1.3 enzymatic identification of pMD19-T-ChIL-2 and pMD19-T-ChIL-18 plasmids

The constructed pMD19-T-ChIL-2 and pMD19-T-ChIL-18 plasmids were double digested with BamHI and SalI, and the results are shown in FIG. 2, with the band sizes consistent with those expected.

3.2 prokaryotic expression and biological Activity of ChIL-2 and ChIL-18 proteins

3.2.1 identification of recombinant expression plasmids pET32a-ChIL-2 and pET28a-ChIL-18

Selecting PstI to carry out enzyme digestion on the constructed recombinant plasmid pET32a-ChIL-2, and setting pET32a as negative control; MluI and SalI are selected to carry out enzyme digestion on the constructed recombinant plasmid pET28a-ChIL-18, pET28a is used as a negative control, the enzyme digestion result is shown in figure 3, and the size of a target band is consistent with the expected size; the sequencing result also shows that the constructed plasmid is correct and has no gene mutation.

3.2.2 inducible expression and purification of ChIL-2 and ChIL-18 proteins

SDS-PAGE results show that after the recombinant bacteria are induced by IPTG, a large amount of expression is carried out at positions of about 27kDa and 23kDa, but the recombinant bacteria without the target gene are not expressed at the positions after the recombinant bacteria are induced, and the recombinant bacteria express ChIL-2 and ChIL-18 proteins. See fig. 4.

Western blot analysis showed specific bands at about 27kDa and 23kDa, confirming that the expressed product is the expected fusion protein, see FIG. 5.

3.2.3 biological Activity of recombinant ChIL-2 and ChIL-18 proteins on splenic lymphocytes of chickens

MTT method results show that the recombinant ChIL-2 and ChIL-18 proteins have obvious lymphocyte proliferation activity, and when the protein concentration of the ChIL-2 is 50 mug/mL, the proliferation index of lymphocytes is 3.3; the proliferation index of lymphocytes is 1.5 when the ChIL-18 protein concentration is 50 μ g/mL.

3.3 eukaryotic expression of ChIL-2 and ChIL-18 genes

3.3.1 identification of pVAX1-ChIL-2 and pVAX1-ChIL-18 eukaryotic expression plasmids

The constructed plasmids pVAX1-ChIL-2 and pVAX1-ChIL-18 were digested with KpnI and XbaI in duplicate, and the results are shown in FIG. 6, with the band sizes corresponding to those expected. The plasmid was sequenced and the sequence was correct.

3.3.2 expression of ChIL-2 and ChIL-18 proteins after transfection of Vero cells with recombinant plasmids

The constructed eukaryotic plasmids pVAX1-cCHIL2 and pVAX1-cCHIL18 are transfected into Vero cells, 3 dishes (with a control) are taken 24h later for indirect immunofluorescence detection, and both ChIL-2 and ChIL-18 proteins are expressed and have visible fluorescence, which is shown in figure 7.

3.4 immune modulatory effects of ChIL-2 and ChIL-18 on NDV-IBV live vaccines

3.4.1 serum NDV antibody levels

Serum is separated after blood collection of 4d, 7d, 14d, 21d, 28d, 35d and 42d of each group of chickens after immunization, the NDV antibody level in the serum is determined, and the result is shown in figure 8, and the NDV antibody level in the serum of each group of chickens 7 days after immunization averagely reaches the immune protection level and can be continued to 42 days after immunization. Feeding ChIL-2 and ChIL-18 did not significantly differ in serum NDV antibody levels compared to the vaccine group alone.

3.4.2 IBV antibody levels in serum

Separating serum after blood collection, and measuring the light absorption value (A) in each reaction hole by using a microplate reader at the wavelength of 450nm, wherein the C.O (critical value) is 0.13+ the average value of the negative control holes, and the A value is positive when the A value is more than or equal to the C.O. The results are shown in FIG. 9.

3.4.3 Effect of ChIL-2 and ChIL-18 on the antiviral challenge of NDV-IBV vaccines

In the vaccine group, 3 of 12 chickens suffered from the disease but did not die, and the protection rate is 75%; 1 chicken in the proteome and the plasmid group respectively have the disease and do not die, and the protection rate is 92%.

3.4.4 Effect of ChIL-2 and ChIL-18 on the antiviral challenge of IBDV polyprotein DNA vaccines

4 of 12 chickens in the group 1 died, and the protection rate was 66%; 4 of 12 chickens in the group 2 died, and the protection rate is high; 3 of 12 chickens in the 3 groups died, and the protection rate is 75%; in 4 groups, 2 of 12 chickens died, and the protection rate is 83 percent; in 5 groups, 3 of 12 chickens died, and the protection rate is 75%; 3 of 12 chickens in 6 groups died, and the protection rate is 75%; in 7 groups, 1 of 12 chickens died, and the protection rate was 92%.

Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above-described embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. a preparation method of recombinant chicken interleukin 2 and interleukin 18 protein, is characterized in that, comprises the steps:

(1) Isolation and culture of SPF chicken spleen lymphocytes;

(2) Extraction of total cell RNA;

(3) RT-PCR amplification of ChIL-2 and ChIL-18 genes;

(4) Cloning and identification of PCR products;

(5) Sequencing analysis of positive clones of ChIL-2 and ChIL-18 genes;

(6) Construction of recombinant expression plasmids pET32a-ChIL-2 and pET28a-ChIL-18;

(7) The recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 were expressed in E. coli.

2. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: the separation and culture of the SPF chicken spleen lymphocyte of described step (1), specifically by blood collection of chicken heart, put to death, The chicken spleen was aseptically taken and placed in cold Hanks solution. After separating the membrane and adipose tissue, it was ground on a 200-mesh copper mesh to make a single-cell suspension, centrifuged at 2000 rpm/min for 15 min, and the pelleted cells were suspended in a solution containing 10% fetal bovine. Serum RPMI1640 medium, take a centrifuge tube, add 4ml of lymphocyte separation solution in advance, slowly add an equal volume of cell suspension to the lymphocyte separation solution, perform density gradient centrifugation, centrifuge at 2000rpm/min for 15min, collect lymphocytes , washed twice with RPMI1640 culture medium, counted the number of viable cells, adjusted the cell concentration to 1×107 cells/ml, added 2ml per well to a 6-well cell culture plate, and placed it in a 40°C, 5%CO2 cell incubator nourish.

3. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: the RT-PCR of described step (2) amplifies ChIL-2 and ChIL-18 gene, specifically respectively in Collect the cultured cells for 6h and 20h, centrifuge at 2000rpm/min at 4°C for 15min, resuspend the cells with 1ml of RPMI1640, and extract total RNA according to the instruction manual of Trizol Reagent: 1×107 cells were vigorously shaken with 1ml of Trizol, centrifuged at 12,000rpm/min at 4°C for 10min, and sucked up Clear to a new Ep tube, add 200 μl of chloroform, shake vigorously, place at 15-30 °C for 2-3 min, centrifuge at 12000 rpm/min at 4 °C for 15 min, carefully pipette the uppermost colorless aqueous phase layer to a new tube, add 1 volume of isopropanol, mix well, centrifuge at 12000rpm/min at 4°C for 10min, discard the supernatant, add 75% ethanol to wash the RNA precipitate, pour off the ethanol, dry in air or vacuum for 10-15min, add 25u1 DEPC-treated water The RNA was dissolved, and the RNA extraction effect was checked by agarose electrophoresis. The extracted total RNA of chicken spleen lymphocytes was stored at -70°C for future use.

4. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: the RT-PCR of described step (3) amplifies ChIL-2 and ChIL-18 gene, specifically comprises 1. primer Design and synthesis, ② RT-PCR amplification of genes;

The primers are designed and synthesized. Specifically, according to the gene sequences of ChIL-2 and ChIL-18 in GeneBank, Primer software is used to design specific primers for amplifying ChIL-2 and ChIL-18. The primers are provided by Shanghai Sangon Bioengineering Co., Ltd. The synthetic primers used to amplify the ChIL-2 and ChIL-18 genes are as follows:

The RT-PCR amplifies the gene, specifically, the dried RNA is dissolved in 15-20 μl of DEPC water, and 11.5 μl of RNA is added to a new PCR tube, and 2 μl of primers are added. ℃ for 5 min, take out the mixture, after ice bath for 1 min, add 2 μl of dNTP, 1 μl of reverse transcriptase, 0.5 μl of RNase inhibitor and 4 μl of 5× Buffer in sequence, put the sample into the PCR machine, 37 ℃ for 1 h, 95 ℃ for 10 min , stored at 4°C or -20°C for later use; the obtained reverse transcribed cDNA product was amplified by Taq enzyme PCR;

The PCR reaction system is as follows: 50 μL

PCR reaction conditions are as follows:

After the reaction, the PCR amplification products were identified by 1% agarose gel electrophoresis, and the DNA fragments were recovered from the gel.

5. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, it is characterized in that: the clone of the PCR product of described step (4) and restriction enzyme digestion identification, it is specifically the DNA fragment clone that will reclaim To the pMD19-T vector, the specific operations are as follows:

The PCR product plus A system is as follows: 10 μL

Ligation of pMD19-T vector: 10 μL

React at 16°C for 30 min or ligate overnight at 4°C to transform DH10B competent cells.

6. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, it is characterized in that: the ChIL-2 of described step (5) and ChIL-18 gene positive clone sequencing analysis, specifically after to be transformed After 16 hours of culture on the plate, pick a single colony into 10 μL of sterile water, take 1 μL of bacterial liquid as a template, use specific primers to carry out colony PCR identification in Taq DNA polymerase system, and transfer positive bacteria to fresh medium. When the liquid was cultured to an appropriate concentration, a small amount of plasmid was extracted, an appropriate restriction site was selected for restriction identification, and the PCR fragment was sequenced.

7. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: the construction of the recombinant expression plasmid pET32a-ChIL-2 and pET28a-ChIL-18 of described step (6), concrete Using pMD19-T-ChIL-2 and pMD19-T-ChIL-18 as templates, the following primers were used for PCR amplification, the target gene fragment was recovered from agarose gel, and pET32a and pET28a were double digested with BamHI and Sall. Fragment ChIL-2 and the vector pET32a, ChIL-18 and the vector pET28a were prepared in a ligation reaction system respectively, ligated overnight at 4°C, and the ligation reaction product was transferred into competent cells DH10B, spread on the plate, and overnight at 37°C. Colony PCR identification, take positive colonies and connect fresh medium, when the bacterial liquid is cultured to an appropriate concentration, carry out a small amount of plasmid extraction. The restriction enzyme sites were selected for restriction identification and sequencing. The primers used to construct pET32a-ChIL-2 and pET28a-ChIL-18 are as follows:

8. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: the recombinant plasmid pET32a-ChIL-2 and pET28a-ChIL-18 of described step (7) are carried out in Escherichia coli Expression, specifically, the recombinant plasmids pET32a-ChIL-2 and pET28a-ChIL-18 were transformed into E. coli BL21, spread on a plate, overnight at 37°C, and a single colony was picked the next day into LB medium, and the concentration of the bacterial solution was A600=1.5 , IPTG was added to a final concentration of 0.1 mmol/L, induced and cultured at 20°C overnight, and the protein was collected.

9. the preparation method of recombinant chicken interleukin 2 and interleukin 18 protein as claimed in claim 1, is characterized in that: also comprises step (8), recombinant ChIL-2 and ChIL-18 protein purification, specifically 10,000g centrifugal 10min collection Bacteria, resuspend the collected cells with 50ml of sonication buffer, sonicate in an ice bath, and centrifuge the lysate at 14,000g for 20min to remove cell debris. The protein purification operation manual was used for purification.

10. The application of chicken interleukin 2 and chicken interleukin 18 protein in combined immunization of SPF chicks with Newcastle disease-infectious bronchitis combined live vaccine.