阅读说明：本技术 鸡球虫多价重组蛋白疫苗及其制备方法和应用 (Chicken coccidiosis multivalent recombinant protein vaccine and preparation method and application thereof ) 是由 刘群 杨旭 陈庆忠 宋星桔 刘晶 安同伟 许建海 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种鸡球虫多价重组蛋白疫苗及其制备方法和应用。所述鸡球虫多价重组蛋白疫苗包含4种鸡球虫蛋白：柔嫩艾美耳球虫棒状体蛋白41,柔嫩艾美耳球虫配子体蛋白22,巨型艾美耳球虫配子体蛋白56,和堆型艾美耳球虫裂殖子蛋白Ea3-1E。将上述4种鸡球虫蛋白分别与大肠杆菌表达载体相连获重组表达载体,表达重组蛋白。将不同种重组蛋白按比例与佐剂混合,免疫母鸡后的后代雏鸡接种球虫后与未免疫母鸡后代雏鸡相比,能够有效提高接种球虫的雏鸡存活率和相对增重,卵囊排出量减少,显示了良好的母源免疫效果。(The invention discloses a chicken coccidiosis multivalent recombinant protein vaccine and a preparation method and application thereof. The chicken coccidia multivalent recombinant protein vaccine comprises 4 chicken coccidian proteins: eimeria tenella rod protein 41, Eimeria tenella gametophyte protein 22, Eimeria maxima gametophyte protein 56, and Eimeria acervulina merozoite protein Ea 3-1E. And (3) respectively connecting the 4 chicken coccidian proteins with an escherichia coli expression vector to obtain a recombinant expression vector, and expressing the recombinant proteins. Different kinds of recombinant proteins are mixed with adjuvants in proportion, and after the offspring chicks immunized with the hens are inoculated with coccidia, compared with the offspring chicks not immunized with the hens, the method can effectively improve the survival rate and the relative weight gain of the chicks inoculated with the coccidia, reduce the oocyst discharge rate, and show good maternal immunization effect.)

1. A chicken coccidia multivalent recombinant protein vaccine, which is characterized by comprising 4 chicken coccidia recombinant proteins: the recombinant protein of the 4 species of the chicken coccidia is prokaryotic expression, sequentially has an amino acid sequence shown as SEQ ID NO.1-4 and is mixed according to the mass ratio of 1:1:1: 1.

2. The chicken coccidia multivalent recombinant protein vaccine of claim 1, wherein the 4 chicken coccidia recombinant proteins have the nucleotide sequences shown as SEQ ID NO.5-8 in sequence.

3. Primer for amplifying the nucleotide sequence according to claim 2, characterized by the following sequence:

amplification of EtROP41 upstream primer:

5’-TTCCAGGGGCCCCTGGGATCCGAACCTCCCCGAGTCAACCT-3’(SEQ ID NO.9)，

amplification of EtROP41 downstream primer:

5’-CACGATGCGGCCGCTCGAGATCCTGGAACTCCCTGGACACC-3’(SEQ ID NO.10)；

amplification of EtGAM22 upstream primer:

5’-CAAGGCCATGGCTGATATCGGCACCTGAGTATCCTTCTCAGCTTG-3’(SEQ ID NO.11)，

amplification of EtGAM22 downstream primer:

5’-TTGTCGACGGAGCTCGAATTGTTGATGTCGGTAGGCTGCTCTTCC-3’(SEQ ID NO.12)；

amplification of EmGAM56 upstream primer:

5’-TTCCAGGGGCCCCTGGGATCCCAGGTTCACCCTTACAGCGAG-3’(SEQ ID NO.13)，

downstream primer for amplification of EmGAM 56:

5’-CGTCTCCGCTCTTTGGCAACCTCGAGCGGCCGCATCGTG-3’(SEQ ID NO.14)；

amplifying Ea3-1E upstream primer:

5’-AGCAAATGGGTCGCGGATCCATGGGTGAAGAGGCTGATAC-3’(SEQ ID NO.15)，

amplifying Ea3-1E downstream primer:

5’-TCGAGTGCGGCCGCAAGCTTGAAGCCGCCCTGGTACAGGT-3’(SEQ ID NO.16)。

4. the preparation method of the chicken coccidiosis multivalent recombinant protein vaccine is characterized by comprising the following steps:

gene amplification: separately performing gene amplification by using the cDNA sequences of Eimeria tenella rod-shaped protein 41, Eimeria tenella gametophyte protein 22, Eimeria maxima gametophyte protein 56 and Eimeria acervulina merozoite protein Ea3-1E as templates, respectively, and using the primers of claim 3;

constructing a recombinant vector: constructing a recombinant vector by using the amplified EtROP41 gene sequence and pGEX-6p-1 skeleton, constructing a recombinant vector by using the amplified EtGAM22 gene sequence and pET-28a skeleton, constructing a recombinant vector by using the amplified EmGAM56 gene sequence and pGEX-6p-1 skeleton, and constructing a recombinant vector by using the amplified Ea3-1E gene sequence and pET-28a skeleton;

recombinant protein expression: respectively transforming the 4 recombinant vectors into expression bacteria, and respectively carrying out induction expression and purification by IPTG; and

mixing: and (4) mixing the 4 chicken coccidian recombinant proteins subjected to induction expression with equal mass to obtain the chicken coccidian recombinant protein.

5. The method for preparing the chicken coccidia multivalent recombinant protein vaccine as claimed in claim 4, wherein the expressing strain is E.coli Transetta (DE3) strain; the induction expression conditions of the Eimeria tenella rod body protein 41, the Eimeria tenella gametophyte protein 22 and the Eimeria acervulina merozoite protein Ea3-1E are all induction at 37 ℃ for 12h, and the induction expression condition of the Eimeria maxima gametophyte protein 56 is induction at 18 ℃ for 18 h.

6. An antibody or serum capable of specifically binding to the chicken coccidia multivalent recombinant protein vaccine of claim 1.

7. The use of the coccidian multivalent recombinant protein vaccine of claim 1 in the maternal immunization of coccidia and the preparation of an anti-coccidia medicament for treating offspring chicks.

8. The use of claim 7, wherein the coccidia multivalent recombinant protein vaccine is used in combination with an adjuvant, and the coccidia multivalent recombinant protein vaccine and the adjuvant are mixed in equal mass.

9. The use of claim 8, wherein the immunization program for gallinaceous maternity immunization comprises:

firstly, the method avoids: mixing the chicken coccidiosis multivalent recombinant protein vaccine with Freund's complete adjuvant in equal mass, and injecting subcutaneously in the breast of a hen of 24-26 weeks old;

and (2) avoiding: carrying out secondary immunization two weeks after the primary immunization, mixing the chicken coccidia multivalent recombinant protein vaccine with Freund incomplete adjuvant in equal mass, and then carrying out subcutaneous injection on the breast of the primary immunized hen; and

and (3) three-step (I): and performing tertiary immunization four weeks after the secondary immunization, mixing the chicken coccidian multivalent recombinant protein vaccine with Freund incomplete adjuvant and other masses, and performing subcutaneous injection on the breast of the hen subjected to the secondary immunization.

10. The use of claim 9, wherein the antibody titer in the serum of the hen is to be detected after the hen is subjected to primary, secondary and tertiary immunization, wherein the detection conditions are as follows: the chicken coccidian multivalent recombinant protein vaccine is coated, the optimal dilution of serum is 1 (350-450), and the optimal dilution of enzyme-labeled antibody is 1: 10000.

Technical Field

The invention relates to the technical field of biological veterinary drugs, in particular to a chicken coccidiosis multivalent recombinant protein vaccine and a preparation method and application thereof.

Background

Chicken Coccidiosis (Coccidiosis) is one of the serious diseases endangering the poultry industry caused by infection with 7 species of Eimeria, and is distributed worldwide. Pathogens that cause coccidiosis in chickens include Eimeria tenella (Eimeria tenella), Eimeria maxima (Eimeria maxima), Eimeria acervulina (Eimeria acervulina), Eimeria brunetti (Eimeria brunetti), Eimeria necatrix (Eimeria necatrix), Eimeria praecox (Eimeria praecox), and Eimeria mitis (Eimeria mitis). Different coccidian parasitize epithelial cells of different intestinal sections of the chicken to cause a series of damages such as enteritis, bleeding, weight gain reduction and even death, and the damage degree is closely related to the coccidian species, the infection amount, the day-old of the chicken and the like. In production, chicken coccidiosis is often mixed infection, the morbidity is up to 50-70%, the mortality is 20-30%, and the mortality is up to 80% in severe cases. There are about 400 million chickens infected with coccidiosis worldwide each year, and the economic loss due to coccidiosis in chickens worldwide is about billions of dollars each year. At present, the prevention and control of chicken coccidiosis mainly depends on anticoccidial drugs and live vaccines, but the use of anticoccidial drugs is limited due to the problems of drug resistance, drug residues and the like; problems with the storage, transport, application and management of live vaccines have limited their widespread use. In addition, already registered in some countriesThe coccidian vaccine is gametophyte protein vaccine of Eimeria maxima, is prepared from extracted natural protein of Eimeria maxima, mainly comprises EmGAM56, EmGAM82 and EmGAM230 proteins in gametophyte stage, and has good maternal immune protection effect. But the manufacturing cost is very high, and the popularization and the application are difficult. Therefore, the development of new products and new methods for preventing and controlling chicken coccidiosis is of great significance.

The key to the development of the chicken coccidian recombinant protein vaccine lies in the screening of protective antigens. Various secreted proteins in the invasion, development and reproduction of coccidia are not only of great significance in their life activities, but also may be important immunogens of coccidia. Therefore, the antigens with better immunogenicity are screened out and effectively combined to form the recombinant protein vaccine. Compared with virulent vaccine, attenuated vaccine and natural protein vaccine, the recombinant protein vaccine has the advantages of high safety, high purity, high stability, high yield and the like, and has a good application prospect.

Maternal immunization (Maternal immunization) of poultry refers to that after the antigen is immunized to a hen, the hen generates specific antibody which can be transmitted to offspring chicks in the form of yolk antibody and provides protection for the chicks within a certain period of time. The advantages of the maternal immunity in the poultry breeding industry are that hundreds of chicks can be well protected by immunizing one hen, and the immunization program is simple and convenient and has low cost.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide a chicken coccidium multivalent recombinant protein vaccine which is a recombinant protein vaccine formed by screening high-quality chicken coccidium antigens and can induce a better immune response of a hen, and a maternal antibody can protect offspring chicks from being infected by chicken coccidium and prevent coccidiosis of the offspring chicks by immunizing the hen.

In order to achieve the above object, the present invention provides a chicken coccidia multivalent recombinant protein vaccine, which comprises 4 chicken coccidia recombinant proteins: eimeria tenella rod-shaped protein 41(EtROP41), Eimeria tenella gametophyte protein 22(EtGAM22), Eimeria maxima gametophyte protein 56(EmGAM56) and Eimeria acervulina merozoite protein Ea3-1E, wherein the 4 chicken recombinant proteins are all in prokaryotic expression, sequentially have amino acid sequences shown in SEQ ID NO.1-4 and are mixed according to the mass ratio of 1:1:1: 1.

The four proteins are obtained by screening in the following modes: screening proteins with high expression in a sporozoite stage from a ToxoDB database through New sera-Genes-Transcriptomics-RNA Seq evaluation, predicting molecular weight, signal peptide, B cell and T cell epitope of the proteins, taking secreted proteins with molecular weight of 20-100kDa, signal peptide, large expression in each stage and more epitope as screening basis, and verifying and screening through a chick immune protection test to obtain the Et.ROP41 protein and Ea3-1E protein. Ea3-1E is an antigen gene which is frequently researched at present and is a surface antigen of E. The Ea3-1E sequence has high homology among E.tenella, E.necatrix and E.acervulina, and can be used as a cross antigen of a candidate vaccine. Rop41 protein is a newly discovered and identified protein; the Etgam22 gene is a multicopy gene specifically expressed by e.tenella at the gametophyte stage, the expression product of which is associated with the oocyst wall formation. Immune protection results show that rEtgam22 can improve weight gain to a certain extent, reduce lesion scores and reduce oocyst yield; the EmGAM56 protein is commercialized abroadThe main components of the coccidian vaccine are verified by laboratories and fields in a plurality of countries and regions, so that the coccidian vaccine has a good immune effect, but the coccidian vaccine is not popularized and used in China due to the problems of complex production process, high cost and the like. Mixing the 4 chicken coccidian recombinant proteins with adjuvant at a certain ratio, and immunizingThe chicken, the serum antibody and the yolk antibody of the hen and the offspring chick attack protection experiment show that the recombinant protein vaccine can induce better immune reaction of the hen, the maternal antibody can protect the offspring chick from being infected by coccidian, and the coccidiosis of the offspring chick can be prevented by immunizing the hen.

In one embodiment of the invention, the 4 chicken coccidian recombinant proteins sequentially have nucleotide sequences shown as SEQ ID No. 5-8.

The invention also provides a primer for amplifying the nucleotide sequence, which has the following sequence: amplification of EtROP41 upstream primer: 5'-TTCCAGGGGCCCCTGGGATCCGAACCTCCCCGAGTCAACCT-3' (SEQ ID NO.9), amplification of the EtROP41 downstream primer: 5'-CACGATGCGGCCGCTCGAGATCCTGGAACTCCCTGGACACC-3' (SEQ ID NO. 10); amplification of EtGAM22 upstream primer: 5'-CAAGGCCATGGCTGATATCGGCACCTGAGTATCCTTCTCAGCTTG-3' (SEQ ID NO.11), amplification of the downstream primer of EtGAM 22: 5'-TTGTCGACGGAGCTCGAATTGTTGATGTCGGTAGGCTGCTCTTCC-3' (SEQ ID NO. 12); amplification of EmGAM56 upstream primer: 5'-TTCCAGGGGCCCCTGGGATCCCAGGTTCACCCTTACAGCGAG-3' (SEQ ID NO.13), downstream primer for amplification of EmGAM 56: 5'-CGTCTCCGCTCTTTGGCAACCTCGAGCGGCCGCATCGTG-3' (SEQ ID NO. 14); amplifying Ea3-1E upstream primer: 5'-AGCAAATGGGTCGCGGATCCATGGGTGAAGAGGCTGATAC-3' (SEQ ID NO.15), and the downstream primer for amplifying Ea 3-1E: 5'-TCGAGTGCGGCCGCAAGCTTGAAGCCGCCCTGGTACAGGT-3' (SEQ ID NO. 16).

The invention also provides a preparation method of the chicken coccidium multivalent recombinant protein vaccine, which comprises the following steps: gene amplification: respectively carrying out gene amplification by using cDNA sequences of Eimeria tenella rod-shaped protein 41(EtROP41), Eimeria tenella gametophyte protein 22(EtGAM22), Eimeria maxima gametophyte protein 56(EmGAM56) and Eimeria acervulina merozoite protein Ea3-1E as templates; constructing a recombinant vector: constructing a recombinant vector by using the amplified EtROP41 gene sequence and pGEX-6p-1 skeleton, constructing a recombinant vector by using the amplified EtGAM22 gene sequence and pET-28a skeleton, constructing a recombinant vector by using the amplified EmGAM56 gene sequence and pGEX-6p-1 skeleton, and constructing a recombinant vector by using the amplified Ea3-1E gene sequence and pET-28a skeletonA body; recombinant protein expression: respectively transforming the 4 recombinant vectors into expression bacteria, inoculating the expression bacteria into 1L of fresh LB (containing AMP 100. mu.g/mL) culture solution to OD of the culture solution_600nmAdding inducer IPTG (0.8mmol/L) after the value is about 1, and respectively carrying out induced expression and purification; and mixing: and (4) mixing the 4 chicken coccidian recombinant proteins subjected to induction expression with equal mass to obtain the chicken coccidian recombinant protein.

In one embodiment of the invention, the expressing bacterium is an e.coli transetta (DE3) strain; the induction expression conditions of the E.tenella rod-shaped body protein 41(EtROP41), the E.tenella gametophyte protein 22(EtGAM22) and the E.acervulina merozoite protein Ea3-1E are all induction at 37 ℃ for 12h (160r/min), and the induction expression condition of the E.maxima gametophyte protein 56(EmGAM56) is induction at 18 ℃ for 18 h.

The invention also provides an antibody or serum capable of being specifically combined with the chicken coccidium multivalent recombinant protein vaccine.

The invention also provides application of the coccidium multivalent recombinant protein vaccine in chicken coccidium maternal immunity and preparation of a medicine for treating offspring chicken coccidium resistance.

In one embodiment of the invention, the chicken coccidia multivalent recombinant protein vaccine and an adjuvant are used together, and the chicken coccidia multivalent recombinant protein vaccine and the adjuvant are mixed in equal mass.

In one embodiment of the present invention, the immunization program for chicken coccidian maternal immunization comprises: firstly, the method avoids: mixing the coccidiosis multivalent recombinant protein vaccine with Freund's complete adjuvant, and injecting subcutaneously into the chest of a hen (hen before egg laying) of 24-26 weeks old; and (2) avoiding: carrying out secondary immunization two weeks after the primary immunization, mixing the chicken coccidia multivalent recombinant protein vaccine with Freund incomplete adjuvant in equal mass, and then carrying out subcutaneous injection on the breast of the primary immunized hen; and exempt from three: and (3) performing tertiary immunization four weeks after the secondary immunization, mixing the chicken coccidia multivalent recombinant protein vaccine with Freund incomplete adjuvant and other qualities, and performing subcutaneous injection on the breast of the hen subjected to the secondary immunization, wherein the immunization dosage of 4 chicken coccidia recombinant proteins in the chicken coccidia multivalent recombinant protein vaccine is 100 mu g/feather.

In an embodiment of the present invention, after the hen is subjected to primary immunization, secondary immunization and tertiary immunization, the antibody titer in the serum of the hen needs to be detected respectively, wherein the detection conditions are as follows: the coccidian multivalent recombinant protein vaccine is coated, the optimal dilution of serum is 1 (350-450), the optimal dilution of enzyme-labeled antibody is 1:10000, and preferably, the coccidian multivalent recombinant protein vaccine is coated after being diluted to 4 mu g/mL.

Compared with the prior art, the invention has the following beneficial effects:

the invention relates to a chicken coccidiosis multivalent recombinant protein vaccine which is formed by combining Eimeria tenella rod body protein 41(EtROP41), Eimeria tenella gametophyte protein 22(EtGAM22), Eimeria maxima gametophyte protein 56(EmGAM56) and Eimeria acervulina merozoite protein Ea3-1E according to the proportion of 1:1:1: 1. The maternal immunity result shows that the specific serum antibody and the yolk antibody of the hen can be maintained for about 12 weeks after the hen is immunized by the recombinant protein. Hatching eggs with high antibody duration are selected to hatch chicks, and offspring chicks of 7-day age and 14-day age are subjected to insect attack protection tests respectively. The serum antibody and the yolk antibody of the hen and the attack protection experiment of the offspring chick show that the recombinant protein vaccine can induce better immune response of the hen, and the maternal antibody can protect the offspring chick from being infected by coccidian, and can prevent coccidiosis of the offspring chick by immunizing the hen.

Drawings

FIG. 1A is an electrophoretic image of an amplification product of EtROP41 gene according to one embodiment of the present invention;

FIG. 1B is an electrophoretic identification chart of the amplification product of EtGAM22 gene according to one embodiment of the present invention;

FIG. 1C is a view showing the electrophoretic identification of the amplification product of Ea3-1E gene according to one embodiment of the present invention;

FIG. 1D is an electrophoretic identification chart of an amplification product of EmGAM56 gene according to an embodiment of the present invention;

FIG. 2A is a SDS-PAGE pattern of the expression and purification of the chicken coccidia recombinant protein EtROP41 according to one embodiment of the invention;

FIG. 2B is a SDS-PAGE pattern of Ea3-1E expression and purification according to one embodiment of the invention;

FIG. 2C is a SDS-PAGE pattern of expression and purification of the chicken coccidian recombinant protein EmGAM56 according to one embodiment of the invention;

FIG. 2D is a SDS-PAGE pattern of expression and purification of the chicken coccidian recombinant protein EtGAM22 according to one embodiment of the invention;

FIG. 3 is a graph of the change in specific serum antibody IgG levels following immunization of breeder hens, in accordance with one embodiment of the present invention.

Description of the main reference numerals:

m, protein Marker; denotes immunization time node.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

The experimental methods involved in the invention are all conventional methods unless otherwise specified.

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

Coli transetta (DE3) strain: purchased from Beijing Quanjin Biotechnology Ltd.

Coli DH5 α: purchased from Beijing, Congress Bio-technology Ltd.

High fidelity enzyme, Taq enzyme: purchased from biotechnology limited of nuozokenza, Nanjing.

Multi-fragment ligation kit, reverse transcription kit: purchased from Beijing Quanjin Biotechnology Ltd.

Nickel affinity chromatography packing: purchased from novagen, usa.

Expression vectors pET-28a (+) and pGEX-6p-1 (+): and (4) storing in a laboratory.

Freund's adjuvant (complete, incomplete): Sigma-aldrich, USA.

24-26 weeks old helenium brown laying hens: purchased from Jinassist farms, Tianjin City.

Eimeria tenella, eimeria maxima and eimeria acervulina: isolated, identified and stored by the national animal parasitic protozoan laboratory of university of agriculture, china.