CN1803195A - Immunity regulating type Eimeria tenella DNA vaccine for preventing and treating chicken coccidiosis - Google Patents

Abstract

The invention provides an immunoregulatory DNA vaccine capable of preventing and treating chicken coccidiosis. This vaccine contains Eimeria tenella (Eimeria tenella) calcium regulatory binding domain protein kinase gene pEtK2 and chicken interleukin-2 gene, and a protease-specific hydrolysis sequence is introduced at the 5' end of the chIL-2 gene. The vaccine contains multiple T cell immune response elements and can effectively prevent and treat chicken coccidiosis.

Description

An Immunomodulatory DNA Vaccine of Eimeria tenella

technical field

The invention relates to the technical field of biological veterinary medicine, and is an immunoregulatory DNA vaccine capable of preventing chicken coccidiosis. This vaccine contains Eimeria tenella (Eimeria tenella) calcium regulation binding domain protein kinase gene pEtK2 and chicken interleukin-2 gene (chIL-2). And a protease-specific hydrolysis sequence was introduced at the 5' end of the chIL-2 gene. This vaccine contains multiple T cell immune response elements.

Background technique

Chicken coccidiosis is an important parasitic disease that harms the poultry industry, and the economic loss caused by coccidiosis is about 2 billion pounds worldwide every year. The disease is caused by 7 species of coccidia of the genus Eimeria, among which E. tenella causes the greatest damage. The current drug control of chicken coccidiosis has brought a series of problems. Such as the emergence of drug resistance, the harm caused by drug residues to the environment, and so on. These issues have attracted increasing attention. People are eager to find an effective way to prevent chicken coccidiosis.

The use of highly toxic vaccines and attenuated coccidia vaccines is a means of prevention, but the highly toxic vaccines are only suitable for breeders, not completely suitable for layer and broiler chickens. Although broiler chickens can use attenuated coccidia vaccines, they have poor stability. The inoculum size is difficult to control, and the cost is high, so that these coccidia vaccines cannot be well developed and utilized. With the rapid development of molecular biology and immunology, people are eager to develop new genetic engineering vaccines by means of genetic engineering to provide new means for the prevention and treatment of chicken coccidiosis.

The immune response of chickens to coccidiosis is the body's comprehensive defense performance against pathogens, including non-specific immunity and specific acquired immunity-humoral immunity and cellular immunity. The humoral immune response is mainly achieved by B lymphocytes dependent on the supraluminal bursa, and the cellular immune response is mainly achieved by T cells dependent on the thymus. It has been found that chicken coccidiosis immunity can be suppressed by removing the thymus, while removing the supraluminal bursa has no effect. It shows that the role of antibodies in the protective immunity of coccidia is small. However, the results of adoptive immune lymphocyte transfer experiments have proved that the T cell immune response is at the core of the coccidia immune response.

Nucleic acid vaccine is a new type of vaccine developed in recent years. Because of its simple operation, it not only has the safety of recombinant subunit vaccine, but also has the advantages of high efficiency and persistence of all-round immune response induced by live attenuated vaccine. One of the hot spots in vaccine research. In recent years, in the aspect of anti-parasitic infection, the research of nucleic acid vaccine has launched active exploration.

The pEtK2 gene was screened from the cDNA library of E. tenella sporozoites by using the antiserum prepared by Dunn in 1996 by using an antigen that has a proliferation effect on lymphocytes, that is, CαEm70/l. The gene size is 1464bp, encoding Calmodulin-binding domain-containing protein kinase of 55KD (EtCDPK, Genebank accession number AY389513). The protein exists in sporozoites and merozoite apical apparatus, can stimulate the proliferation of T lymphocytes in infected chickens, and can provide better protection for infected chickens.

Cytokines are a group of immunoregulatory factors with a wide range of biological activities produced by immune cells or non-immune cells in the body. They can activate and regulate immune active cells in vivo and play an important role in the generation and regulation of immune responses. The main function of IL-2 is to promote the differentiation and proliferation of CD8 ⁺ T cells and CD4 ⁺ T cells, maintain the growth of T cells, and promote the functions of natural killer cells (NK) and B cells. Therefore, it is also called T cell growth factor (TCGF). The role of IL-2 in anti-E.tenella and anti-E.acervμlina infection has been confirmed. Gaarter et al. (Caarter LL. et al., Regulation of T cell subsets frommnative to memory. J Immunol. 1998, 21 (3): 181-187) reported that injecting IL-2 to chickens on the 3rd and 5th days of infection could Reduce the output of oocysts. Li Xiangrui et al. (Li Xiangrui, Jin Hong, Lu Jingliang. Cloning of Chicken Interleukin-2 cDNA. Journal of Nanjing Agricultural University, 1999, 22(2): 80-84) have cloned and expressed chicken IL-2 gene.

Contents of the invention

The invention provides an immunoregulatory DNA vaccine capable of preventing or treating chicken coccidiosis.

The invention provides a method for preparing an immunoregulatory DNA vaccine capable of preventing or treating chicken coccidiosis.

In addition, the present invention also provides the application of the DNA vaccine.

The present invention is based in part on the inventor's discovery that a eukaryotic plasmid vector containing the protein kinase gene pEtK2 of the Eimeria tenella (E. tenella) calcium-binding domain protein kinase can provide chickens with Effective immune protection against coccidia; containing chIL-2 can increase the leaching level of cecal tonsil and spleen in chickens infected with Eimeria coccidia (E.tenella) and the induction of chIL-2, and can reduce the lesion score and The number of oocysts in the feces; when preparing DNA vaccines, introducing the chicken interleukin-2 gene together with the coccidia protective gene (calmodulin binding domain protein kinase gene pEtK2) into a eukaryotic plasmid can further enhance the coccidia protective gene immune protection effect.

In one aspect, the present invention provides an immunomodulatory DNA vaccine capable of preventing or treating chicken coccidiosis, the vaccine comprising an Eimeria tenella (E.tenella) calcium-binding domain protein kinase gene inserted therein Eukaryotic plasmid vector of pEtK2.

The DNA vaccine of the present invention uses a eukaryotic plasmid expression vector as a structural frame, and the eukaryotic plasmid vector not only has the basic elements of the expression vector, but also inserts a target gene at a restriction enzyme cutting site. Any eukaryotic plasmid vector known in the art can be used in the present invention, for example, including but not limited to pcDNA3.0, pcDNA3.1, pVAX1.0 and pcDNA4/His Max. In a specific preferred embodiment of the present invention, the eukaryotic plasmid vector is pcDNA4/His Max (purchased from Invitrogen, USA).

The immunogenic pEtK2 gene used in the present invention is a coccidial protective antigen gene with a size of 1464bp, which is an antiserum prepared using CαEm70/1 antiserum (an antigen that has a proliferative effect on lymphocytes). serum), screened from the E. tenella sporozoite cDNA library. The protein encoded by the gene exists in sporozoites and merozoite apical apparatus, can stimulate the proliferation of T lymphocytes in infected chickens, and can provide good protection for infected chickens.

In a preferred embodiment of the present invention, the vaccine further comprises the chicken interleukin-2 gene (chIL-2) which is connected together with the gene pEtK2 and inserted into the eukaryotic plasmid vector to enhance the DNA vaccine. Immunogenicity.

The inventor successfully cloned and expressed the chicken IL-2 gene (chIL-2), the nucleotide sequence of which is shown in positions 1483-1905 in SEQ ID NO:1. The present inventor further studied the effect of chicken IL-2 on the cellular immunity level of chicken Eimeria tenella. In short, the test set up the control group, different doses of oocyst infection group and different doses of oocyst infection plus interleukin-2 (IL-2) group. The worms were challenged on the basis of the first infection. The lymphocyte level of cecal tonsil and spleen lymphocytes at different times after the worm was detected, and the results were analyzed in combination with cecal lesion score and fecal oocyst count. The results showed that after the first infection, lymphocyte transformation level of cecum tonsil in simple infection group was continuously inhibited; lymphocyte transformation level of spleen in each oocyst infection group decreased on the 4th day. Injection of IL-2 at the same time as the infection made the level of cecal tonsil lymphoma in the 1, 20, and 50,000 oocyst infection groups higher than that of the simple infection group on the 4th, 6th, and 9th day respectively; levels have risen significantly. After challenge with worms, the levels of cecum tonsil and spleen lymphoma in different infection groups showed a downward trend. Adding IL-2 can increase the transformation level of cecal tonsil lymphocytes, but there is no dose difference; the transformation level of spleen lymphocytes is higher than that of the simple infection group, and higher than that of the control group on the 9th day. The results of this experiment showed that: 1) Adding recombinant chicken IL-2 can significantly reduce the lesion score and excretion of oocysts in the feces of the 10,000 and 20,000 oocyst infection groups after the first infection and challenge; 3) Recombinant IL-2 can improve the transformation level of lymphocytes in the cecal tonsils and spleen of the first infection, thereby improving the spleen and cecal lymphocytes caused by coccidia infection for the first time. and cecal tonsil immunosuppression; 4). Recombinant IL-2 can increase the transformation level of cecal tonsil and spleen lymphocytes after challenge, and has an immune protection effect on challenge worms.

Based on the above findings, therefore in one embodiment of the present invention, the chicken interleukin-2 gene (chIL-2 gene (chIL- 2), it was found that the immune protection effect of the DNA vaccine was further enhanced.

In a particularly preferred embodiment of the present invention, the vaccine further comprises a nucleotide sequence encoding a protease coagulation factor Xa _- specific hydrolysis sequence CCT ACC CTC GAT inserted between the gene pEtK2 and the gene chIL-2 . Insert the protease-specific hydrolysis sequence coding nucleotide sequence between the coccidia protective gene and chIL-2, so that the coccidia protective antigen expressed in the DNA vaccine of the present invention in muscle cells or antigen-presenting cells is compatible with The fusion protein of chIL-2 contains a protease-specific site. Therefore, it can be ensured that the protease can hydrolyze the fusion protein at the specific site of the protease to obtain the coccidial antigen and chIL-2 protein, so that these two proteins can independently form a high-level structure and exert corresponding functions.

In a preferred embodiment of the present invention, the 5' end of the chIL-2 gene is introduced into the 5' end of the chIL-2 gene by using _the nucleotide sequence encoding nucleotide sequence GAATTCCCTACCCTCGAT (such as SEQ ID NO: 1 1465-1482 nucleotides in ), thus preparing a fusion gene pEtK2-X _a -chIL-2 inserted into the eukaryotic plasmid vector (according to the direction from 5' to 3'), the fusion gene It has the nucleotide sequence shown in SEQ ID NO:1.

In a second aspect of the present invention, a method for producing an immunomodulatory DNA vaccine of Eimeria tenella is provided. In short, its technical route is as follows:

1. Preparation of coccidian protective antigen gene pEtK2 and chicken interleukin 2 gene (chIL-2) by PCR amplification

The genes pEtK2 and chIL-2 were cloned by means of PCR amplification. Wherein in a specific preferred embodiment of the present invention, by means of primer design, a section of protease blood coagulation factor Xa specific hydrolysis sequence encoding nucleotide sequence GAA TTC CCTACC CTC GAT is introduced at the 5' end of the chIL-2 gene;

2. Construction of DNA vaccine recombinant eukaryotic plasmid and corresponding genetically engineered bacteria

Utilize the routine recombination method of molecular biology, prepare the recombinant eukaryotic plasmid containing pEtK2 or containing pEtK2-(Xa)-chIL-2, then use it to transform the host bacterium, in a technical scheme of the present invention, the host bacterium It is Escherichia coli JM109, and the recombinant genetically engineered bacteria were obtained after screening;

3. Fermentation of genetically engineered bacteria and large-scale separation and purification of DNA vaccine recombinant eukaryotic plasmids.

The third aspect of the present invention is the use of the Eimeria tenella (Eimeriatenella) immunomodulatory DNA vaccine described in the present invention. After direct immunization of animals with purified recombinant DNA vaccines containing pEtK2 or pEtK2-(Xa)-chIL-2, it was found through multiple test indicators such as feces index per gram, oocyst reduction rate, cecal lesion score and coccidial index. , both of which have good immune protection effects (the latter has a more significant protective effect). Therefore, the DNA vaccine of the invention can be used to prepare pharmaceutical preparations that can prevent or treat chicken coccidiosis.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the Eimeria tenella immunoregulatory DNA vaccine of the present invention.

Figure 2 is a schematic diagram of the structure of the recombinant plasmid pBV220-chIL-2 encoding chicken interleukin 2 constructed in our laboratory.

Fig. 3 is a flowchart of the construction of the recombinant plasmid pMD18-T-pEtK2.

Fig. 4 is a flowchart of the construction of the recombinant plasmid pET-chIL-2.

Figure 5 is a flow chart of the construction of the recombinant plasmid pcDNA4/His Max-pEtK2.

Figure 6 is a flow chart of the construction of the recombinant plasmid pcDNA4/His Max-pEtK2-Xa-chIL-2.

Fig. 7 is RT-PCR detection DNA vaccine pcDNA4/His Max-pEtK2, pVAX1.0-pEtK2,

Transcription of pcDNA4/His Max-pEtK2-chIL-2 and pVAX 1.0-pEtK2-chIL-2 in chicken muscle cells, RT-PCR product agarose gel electrophoresis.

1 is DL2000 Marker; 2 and 3 are RT-PCR amplification products of pcDNA4/His Max-pEtK2 and pVAX 1.0-pEtK2 recombinant plasmid injection site muscle (breast muscle) (amplified with pEtK2 primers); 4 and 5 are pcDNA4/His Max-pEtK2-chIL-2 and pVAX1.0-pEtK2-chIL-2 recombinant plasmid injection site muscle (pectoral muscle) RT-PCR amplification products (amplified with pEtK2 primers); 6 and 7 are pcDNA4/His Max-pEtK2 RT-PCR amplification products (amplified with IL-2 primers) of the muscle (pectoral muscle) at the injection site of recombinant plasmids of -Xa-chIL-2 and pVAX1.0-pEtK2-Xa-chIL-28, 9, 10 and 11, respectively RT for pcDNA4/His Max-pEtK2, pVAX1.0-pEtK2, pcDNA4/His Max-pEtK2-Xa-chIL-2 and pVAX1.0-pEtK2-Xa-chIL-2 recombinant plasmid non-injected muscle (leg) -PCR amplification products (amplified with pEtK2 primers); 12 and 13 are the RTs of pcDNA4/His Max-pEtK2-chIL-2 and pVAX1.0-pEtK2-chIL-2 recombinant plasmids, respectively, of the non-injection site muscles (legs) - PCR amplification product (amplified with chIL-2 primers).

Fig. 8 is a graph showing the expression results of DNA vaccines containing pEtK2 or pEtK2-(Xa)-chIL-2 in muscle detected by Western-blotting.

1 and 2 are the protein expression results of Western-blotting detection of pcDNA4/His Max-pEtK2 and pVAX1.0-pEtK2 recombinant plasmids in non-injected muscle (leg); 3 and 4 are Western-blotting detection of pcDNA4/His Max-pEtK2- Protein expression results of chIL-2 and pVAX1.0-pEtK2-chIL-2 recombinant plasmid non-injected muscle (leg); 5 and 6 are Western-blotting detection of pcDNA4/His Max-pEtK2-Xa-chIL-2 and pVAX1 .0-pEtK2-Xa-chIL-2 recombinant plasmid injection site muscle (breast muscle) protein expression results; 7 and 8 are Western-blotting detection pcDNA4/His Max-pEtK2 and pVAX1.0-pEtK2 recombinant plasmid injection site muscle (breast muscle ) protein expression results.

Fig. 9 is the pVAX1.0-pEtK2-Xa-chIL-2 vaccine immunized group, the cecal lesions of chickens after challenge.

Fig. 10 is a picture of cecal lesions of chickens in the PBS immunized group after challenge.

Example

Basic materials: recombinant plasmid pBV220-chIL-2 containing chicken interleukin-2 gene (chIL-2) (constructed in our laboratory, see accompanying drawing 2 for structure); eukaryotic expression vector pcDNA4/His Max and pVAX1.0 ( U.S. Invitrogen Company product); pMD18-T (Dalian Bao Biological (TaKaRa) Engineering Co., Ltd.) pET-28 (+) (U.S. Novagen Company product).

Preparation of embodiment 1.pEtK2 gene:

1. Synthesize primers P1 and P2, the sequences are:

The following primers were designed according to the published nucleotide sequence of pEtK2 (Genebank accession number AY389513):

P1: 5'-AA GGATCC CCAGCAGCGTCCAA-3′

P2: 5'-GAA GAATTC CGCTGCGGTATCTCCG-3'

The underlined parts are the introduced restriction sites BamHI and EcoRI, respectively; the boxed part is the initiation codon.

2. Extraction of coccidia total RNA:

Take 0.1 g (about 107) of pure sporulated oocysts of E. tenella and put them in a glass homogenizer, add 1ml Trizol reagent, grind for 5 minutes to rupture the oocyst wall, and extract the total sporozoite RNA in one step. The specific steps are as follows: put the homogenized solution into a 1.5ml eppendorf tube, add 0.2mL of 24:1 chloroform/isoamyl alcohol mixture, shake the sample vigorously for 15s, put it on ice for 5min, centrifuge at 12000 rpm at 4°C for 15min, carefully remove the Transfer the aqueous phase (upper layer) of RNA to another eppendorf tube and put it on ice, add 0.5ml of isopropanol, mix the sample gently and leave it at room temperature for 10 minutes, centrifuge at 12000 rpm at 4°C for 15 minutes, and the RNA will form a yellow-white color at the bottom of the test tube Precipitate, remove isopropanol carefully, add 1ml 70% ethanol to wash RNA pellet, resuspend RNA pellet by shaking or pipetting, centrifuge at 12000 rpm at 4°C for 8min, remove ethanol, dry at room temperature, measure A260/A280 ratio, add 20μl DEPC The treated water was stored at -20°C.

3. Synthesis of cDNA

Add reagents in the following proportions to synthesize the first strand of cDNA:

Total RNA 10μl

500μg/ml Oligo(dT) 1.0μl

After mixing the above reagents, place it at 70°C for 5 minutes, immediately put it on ice to cool, and then add the following ingredients:

2.5mmol/L dNTP 3.5μl

RNASin (TaKaRa company) 1.0μl

5×RT buffer 5.0μl

200U/μl M-MLV 1.5μl

25mmol/L MgCl ₂ 23.0μl

Make up to 25.0 μl with RNase-free water, mix well, centrifuge briefly at low speed, shake off the droplets on the tube wall, and react at 37°C for 1 hour. Inactivate reverse transcriptase at 90°C for 5 minutes. Reverse transcription products were immediately subjected to PCR or stored at -20°C for later use.

4. PCR amplification of pEtK2 gene

Add the following components to a thin-walled PCR tube for PCR amplification:

cDNA 2.5μl

2.5mmol/L dNTP 4.0μl

P1(50pmol) 1.0μl

P2(50pmol) 1.0μl

10×PCR buffer 5.0μl

25mmol/L MgCl ₂ 23.0μl

Taq DNA polymerase (5U/μl) 0.5μl

wxya ₂ O 33.0μl

Total 50.0μl

After centrifuging and mixing the above components, denature on a PCR machine at 94°C for 5 min; 94°C for 45 sec, 60°C for 60 sec, 72°C for 75 sec, a total of 30 cycles; then extend at 72°C for 10 min.

Sequencing shows that the amplified pEtK2 gene has the nucleotide sequence shown in the 1-1464th position in SEQ ID NO:1.

Example 2. Preparation of chIL-2 gene:

1. Synthesize primers P3 and P4, the sequences are:

TGCAAAGTACTGATCT-3’P3: 5'-CTA GAATTC CCTACCCTCGAT

TGCAAAGTACTGATCT-3'

P4: 5'- TTAGTCGACTTGCAGATATCTCACAAAGTT -3'

Wherein the underlined part is the introduced enzyme cutting site EcoRI and SalI; the square part is the initiation codon; the italicized letter part is the coagulation factor _Xa specific hydrolysis sequence encoding nucleotide sequence (such as the 1465-th in SEQID NO: 1 The nucleotide sequence shown at position 1482).

2. PCR amplification of chIL-2 gene

Add the following components to a thin-walled PCR tube for PCR amplification:

Plasmid pBV220-chIL-2(50ng/μl) 1.0μl

2.5mmol/L dNTP 4.0μl

P3(50pmol) 1.0μl

P4(50pmol) 1.0μl

10×PCR buffer 5.0μl

25mmol/L MgCl ₂ 23.0μl

Taq DNA polymerase (5U/μl) 0.5μl

wxya ₂ O 34.5μl

Total 50.0μl

After centrifuging and mixing the above components, denature at 94°C for 5 minutes on a PCR instrument; 30 cycles at 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 45 seconds; then extend at 72°C for 10 minutes.

Since P3 and P4 were used as primers, the 5' end of the resulting chIL-2 gene contained coagulation factor Xa-specific hydrolysis sequence-encoding nucleotide sequence (i.e., Xa-chIL-2)

Sequencing shows that the amplified chIL-2 gene has the nucleotide sequence shown in the 1483-1905th positions in SEQ ID NO:1.

The preparation of embodiment 3.DNA vaccine

1. Construction of recombinant plasmid pMD18-T-pEtK2 (see Figure 3)

Get 50 μ l of the PCR product obtained in Example 1, electrophoresis on 1% agarose gel, cut out the agarose gel at the band of interest under ultraviolet light, reclaim and purify the fragment of interest with the gel recovery kit of Dalian Bao Biological Company, method Refer to the instruction manual. Take the purified PCR product and connect it with the pMD18-T vector linearized after digestion with BamHI and EcoRI. The reaction system is as follows:

PCR recovery product 4.0μl

pMD18-T vector 1.0μl

Ligation solution I 5.0μl

Total volume 10.0μl

The above components were mixed in a thin-walled eppendorf tube and connected overnight at 4°C. The ligation product was transformed into competent Escherichia coli JM109, the plasmid was extracted, and identified by double enzyme digestion and sequencing with BamHI and EcoRI, and it was determined to be pMD18-T-pEtK2.

2. Construction of recombinant plasmid pET-Xa-chIL-2 (see Figure 4)

Get 50 μ l of the PCR product obtained in Example 2, electrophoresis on 1% agarose gel, cut out the agarose gel at the band of interest under ultraviolet light, and reclaim and purify the target fragment with the gel recovery kit of Dalian Bao Biological Company. Refer to the instruction manual. Get the purified PCR product and plasmid pET-28(+), use EcoRI and Sal I double digestion respectively, and the product is purified and recovered. The two purified products were then ligated with T4 DNA ligase. The connection reaction system is as follows:

PCR product/EcoR I+Sal I (20ng/μl) 5.0μl

pET-28(+)/EcoRI+Sal I(25ng/μl) 2.0μl

5×T4 connection buffer 2.0μl

T4DNA Ligase (2U/μl) 1.0μl

Total volume 10.0μl

The above components were mixed in a thin-walled eppendorf tube and connected overnight at 4°C. The ligation product was transformed into competent Escherichia coli BL21, the plasmid was extracted, and identified by EcoRI and SalI double enzyme digestion and sequencing, and it was determined to be pET-Xa-chIL-2.

3. Construction of recombinant plasmid pVAX1.0-pEtK2 (or pcDNA4/HisMax-pEtK2) (see Figure 5)

Use BamHI and EcoRI to double-digest and clone the plasmid vectors pMD18-T-pEtK2 and pVAX1.0 (or pcDNA4/HisMax), respectively, recover the pEtK2 target gene and pVAX1.0 (or pcDNA4/HisMax) large fragments, and connect them according to the appropriate ratio , the ligation product was transformed into competent Escherichia coli JM109, the plasmid was extracted, identified by double digestion with BamHI and EcoRI, and determined to be pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) DNA vaccine.

4. Construction of the recombinant plasmid pcDNA-pEtK2-Xa-chIL-2 (see Figure 6)

Use EcoRI+NotI double digestion to clone plasmid vectors pET-Xa-chIL-2 and pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2), then recover the Xa-chIL-2 target gene and pVAX1.0-pEtK2 ( or pcDNA4/His Max-pEtK2) large fragments were ligated according to an appropriate ratio, the ligated product was transformed into competent Escherichia coli JM109, the plasmid was extracted, identified by double enzyme digestion with EcoRI and NotI, and determined to be pVAX1.0-pEtK2-Xa-chIL- 2 (pcDNA4/His Max-pEtK2-Xa-chIL-2) DNA vaccine.

Example 4. Detection of expression of immune-modulating DNA vaccines in chickens

A large number of pVAX1.0-pEtK2 (or pcDNA4/His Max-pEtK2) and pVAX1.0-pEtK2-Xa-chIL-2 (pcDNA4/His Max-pEtK2-Xa-chIL-2) recombinant plasmids were extracted respectively, and injected intramuscularly in the chest 100 μg/piece of 14-day-old chicks, take the injection site (breast muscle) and non-injection site muscle (leg) after 7 days

1. RT-PCR detection of DNA vaccine transcription

Muscle total RNA was extracted by one-step method, and pEtK2 gene and chIL-2 gene were respectively amplified by RT-PCR. The results showed that the DNA vaccine was transcribed in chicken muscle cells (see Figure 7).

2. Western-blotting detection of DNA vaccine translation

Take samples from the injection site (chest muscle) and non-injection site muscle (leg) respectively, transfer to nitrocellulose membrane after SDS-PAGE electrophoresis, ponceau red staining, add 5% skimmed milk powder after washing to block irrelevant protein binding site 1 Then add anti-pEtK2 recombinant protein immune rabbit serum (primary antibody) to act for 1 hour, add HRP-labeled goat anti-rabbit antibody (secondary antibody) to act for 1 hour after washing with washing buffer, and finally develop color with DAB chromogenic solution. It was found that the DNA vaccine was well expressed in chicken muscle cells (see Figure 8)

Embodiment 5. Animal experiment result of DNA vaccine of the present invention

120 1-day-old chicks were randomly divided into 4 groups, 30 in each group. 100 μl PBS (first group), 100 μl PBS (second group), 100 μg pVAX 1.0-pEtK2 (third group), 100 μg pVAX 1.0-pEtK2-Xa-chIL-2 ( Fourth group). At the age of 28 days, 0 sporulated oocysts of E. tenella (group 1), 10 ⁵ (group 2), 10 ⁵ (group 3) and 10 ⁵ (group 4) were orally infected respectively. Statistical analysis was performed on the immune protection effect.

1. Weight gain effect

The chickens in each group were weighed one by one before the injection of the DNA vaccine, and then the corresponding chickens were weighed one by one before and on the 7th day after the challenge, and the weight changes of the chickens before and after the challenge were observed, and then calculated Average and relative weight gain. Average weight gain 1 = weight when attacking worms - weight when first immunization; average weight gain 2 = weight when last culling - weight when attacking worms; relative weight gain = (average weight gain of each test group/average gain of non-infected and non-immune groups Weight) × 100%. The weight gain result (seeing table 1) shows that the injection of the vaccine has no effect on the weight gain of the chicken, that is, the toxic and side effects of the vaccine are equivalent to PBS. However, the pVAX 1.0-pEtK2-Xa-chIL-2 DNA vaccine immunized group had no significant difference in weight gain from the non-infected non-immune group after challenge, indicating that pVAX 1.0-pEtK2-Xa-chIL-2 DNA vaccine had obvious protective effect.

Table 1: The weight gain effect of the DNA vaccine of the present invention group average weight gain 1 Relative weight gain 1(%) average weight gain 2 Relative weight gain 2(%) Non-infected non-immune group (first group) Infected non-immune group (second group) pVAX1.0-pEtK2 (third group) pVAX1.0-pEtK2-Xa-chIL-2 (fourth group) 66.30±12.58 ^a 56.92±8.28 ^a 62.52±12.66 ^a 66.75±9.36 ^a 100.0085.8594.30100.83 220.15±19.59 ^a 99.23±25.66 ^c 187.90±15.87 ^b 215.30±21.11 ^a 10045.085.3597.80 The same letter in the same column means no significant difference, no same letter means significant difference, and different lowercase letters means significant difference (p<0.05).

2. The number of oocysts per gram of feces

Check the feces every day after attacking the worms. Collect the feces of each group on the seventh day. After mixing, take 10g of each group and add 90mL distilled water to make a 10-fold dilution. Count the number of eggs per gram of feces.

Table 2. Number of oocysts per gram of feces group Number of oocysts per gram of feces × 10 ⁶ Non-infected non-immune group (first group) Infected non-immune group (second group) pVAX1.0-pEtK2 recombinant plasmid (third group) pVAX1.0-pEtK2-Xa-chIL-2 recombinant plasmid (fourth group) 04.680.820.710

3. Oocyst reduction rate

The reduction rate of oocysts is another indicator for judging the protective effect of the vaccine, and its calculation formula is as follows: reduction rate of oocysts = [(number of excreted fecal oocysts in the infected non-immune group - excreted fecal oocysts in the test group)/feces in the infected non-immune group Oocyst discharge number]×100%.

Table 3: Oocyst reduction rate group Oocyst reduction rate Non-infected non-immune group (first group) Infected non-immune group (second group) pVAX1.0-pEtK2 recombinant plasmid (third group) pVAX1.0-pEtK2-Xa-chIL-2 recombinant plasmid (fourth group) 100% 0% 82.47% 84.83%

4. Cecal lesion score

On the 7th day after attacking the worms, 10 chickens from each group were dissected to observe cecal lesions. Cecal lesions were scored according to the Johnson method. That is to say, cecal lesions are divided into five grades from 0 to +4. 0 points: normal, no gross lesions; +1 points: there are a few scattered petechiae on the cecum wall, the intestinal wall is not thickened, and the contents are normal; +2 points: the number of lesions is large, and the contents of the cecum are obviously bloody , the cecum wall is slightly thickened, and the content is normal; +3 points: there is a lot of blood or cecum core (blood clot or off-white cheese-like banana-shaped lump) in the cecum, the cecum wall is obviously thick, and the feces content in the cecum is small; +4 points: Enlarged cecum due to a large amount of blood or intestine core, with or without feces in the intestine core, and +4 points for dead chickens. When the cecal lesions on both sides are inconsistent, the more serious side shall prevail. Relative lesion score = (lesion score of the experimental group/lesion score of the infected non-immune group) × 100% (see Figures 9 and 10 for the lesions).

Table 4. Cecal lesion score group Cecal Lesion Score Non-infected non-immune group (first group) Infected non-immune group (second group) pVAX1.0-pEtK2 recombinant plasmid (third group) pVAX1.0-pEtK2-Xa-chIL-2 recombinant plasmid (fourth group) 0±0 ^a 3.60±0.46 ^c 1.30±0.53 ^ab 0.40±0.31 ^ab

Note: The same letter in the same column means no significant difference, and no same letter means significant difference,

Different lowercase letters represent significant differences (p<0.05).

5. Anti-coccidial index (ACI)

ACI is a number of parameters including survival rate, weight gain, lesions, oocyst production, etc. After comprehensive evaluation, it can be used as an indicator to determine coccidia drug resistance or drug efficacy, and can also be used as an indicator to detect the protective effect of coccidial vaccines. ACI determination formula: ACI = (survival rate + relative weight gain rate) - (lesion value + oocyst value). (ACI ≥ 180 means the protective effect is obvious, ACI = 160-179 means the protective effect is average, ACI < 160 means no protective effect.)

The results are shown in Table 5. The pVAX1.0-pEtK2-Xa-chIL-2 DNA vaccine of the present invention has an ACI of 198.20 and has a good immune protection effect.

Table 5: Anti-coccidial index (ACI) group survival rate relative weight gain lesion value Oocyst value ACI Non-infected non-immune (group 1) infected non-immune (group 2) pVAX1.0-pEtK2 (group 3) pVAX1.0-pEtK2-Xa-chIL-2 (group 4) 100100100100 10045.0785.3597.8 036134 0111 200.00108.07161.35192.80

Use pcDNA4/His Max-pEtK2 and pcDNA4/His Max-pEtK2-Xa-chIL-2 to replace pVAX1.0-pEtK2 and pVAX1.0-pEtK2-Xa-chIL-2, respectively, and carry out the above experiments (other controls, steps and parameters are exactly the same), and also achieved satisfactory approximate results (data not shown).

Sequence Listing

<110> Nanjing Agricultural University

<120>An Immunomodulatory DNA Vaccine of Eimeria tenella

<210>1

<211>1905

<212>DNA

<213> Artificial sequence

<220>

<223> fusion gene

<220>

<221> CDS (calmodulin binding domain protein kinase gene pEtK2)

<222>(1)..(1464)

<220>

<221> coding sequence of protease coagulation factor X _a specific hydrolysis sequence

<222>(1465)..(1482)

<220>

<221> CDS (chicken interleukin-2 gene)

<222>(1483)..(1905)

<400>1

atg cca gca gcg tcc aag tca gac aaa ctg gct gcc acg ccg ggc atg 48

Met Pro Ala Ala Ser Lys Ser Asp Lys Leu Ala Ala Thr Pro Gly Met

1 5 10 15

ttt gtg cag cac tct aca gca gcc ttc agc gat agg tac aag ggc cag 96

Phe Val Gln His Ser Thr Ala Ala Phe Ser Asp Arg Tyr Lys Gly Gln

20 25 30

cgc gtg ttg ggc aag ggc agt ttc ggt gaa gtt att ttg tgc aaa gat 144

Arg Val Leu Gly Lys Gly Ser Phe Gly Glu Val Ile Leu Cys Lys Asp

35 40 45

aaa gta act gga cag gaa tat gcc gtg aag gtg att tca aaa cgg caa 192

Lys Val Thr Gly Gln Glu Tyr Ala Val Lys Val Ile Ser Lys Arg Gln

50 55 60

gtg aag cag aag acg gac aaa gag ctg ctg ctc aag gag gtg gag ctc 240

Val Lys Gln Lys Thr Asp Lys Glu Leu Leu Leu Lys Glu Val Glu Leu

65 70 75 80

ctt aag aag ctg gtc cac ccc aac atc atg aag ctc tac gag ttc ttc 288

Leu Lys Lys Leu Val His Pro Asn Ile Met Lys Leu Tyr Glu Phe Phe

85 90 95

gag gac aag ggc tac ttc tac tta gtc aca gaa gtc tat acc ggc gga 336

Glu Asp Lys Gly Tyr Phe Tyr Leu Val Thr Glu Val Tyr Thr Gly Gly

100 105 110

gag ctc ttc ggc gag atc atc agc aga aag agg ttc agc gag gtt gac 384

Glu Leu Phe Gly Glu Ile Ile Ser Arg Lys Arg Phe Ser Glu Val Asp

115 120 125

gct gcc cgc atc att cga caa gtg ctt tcg ggc att acg tat atg cac 432

Ala Ala Arg Ile Ile Arg Gln Val Leu Ser Gly Ile Thr Tyr Met His

130 135 140

aag aac aaa atc gtt cac aga gac ctc aag cct gaa aat ctg ctg ctg 480

Lys Asn Lys Ile Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu

145 150 155 160

gaa aac aaa aga aaa gat gct aac att cgc atc att gac ttc ggc ctt 528

Glu Asn Lys Arg Lys Asp Ala Asn Ile Arg Ile Ile Asp Phe Gly Leu

165 170 175

tcc act cac ttt gag tcc acc aag gaa atg aag gac aag atc gga acg 576

Ser Thr His Phe Glu Ser Thr Lys Glu Met Lys Asp Lys Ile Gly Thr

180 185 190

gcc tat tac att gcc cca gag gtc ttg cat ggg gcc tac gac gag aag 624

Ala Tyr Tyr Ile Ala Pro Glu Val Leu His Gly Ala Tyr Asp Glu Lys

195 200 205

tgc gac gtg tgg tcc aca ggg gtc att ttg tat atc ctt ctt tct gga 672

Cys Asp Val Trp Ser Thr Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly

210 215 220

tgc ccc cca ttt aac ggg gca aac gaa ttt gac att ttg aag aaa gtc 720

Cys Pro Pro Phe Asn Gly Ala Asn Glu Phe Asp Ile Leu Lys Lys Val

225 230 235 240

gaa aaa gga aag ttc acc ttg gat ctg ccg caa tgg aag aag gtg agc 768

Glu Lys Gly Lys Phe Thr Leu Asp Leu Pro Gln Trp Lys Lys Val Ser

245 250 255

gag tca gcc aaa gac ttg att cgt aag atg ctg gcg tac gtg ccc aca 816

Glu Ser Ala Lys Asp Leu Ile Arg Lys Met Leu Ala Tyr Val Pro Thr

260 265 270

atg cgg ata tct gca cga gac gcc ctc gag cac gag tgg ctc aag act 864

Met Arg Ile Ser Ala Arg Asp Ala Leu Glu His Glu Trp Leu Lys Thr

275 280 285

aca gac gca gcc act gac agt att gat gtg cct tcg ctt gag agc acc 912

Thr Asp AlaAla Thr Asp Ser Ile Asp Val Pro Ser Leu Glu Ser Thr

290 295 300

ata ctt aac atc aga cag ttc cag ggg acg caa aag ctt gcc gcg gct 960

Ile Leu Asn Ile Arg Gln Phe Gln Gly Thr Gln Lys Leu Ala Ala Ala

305 310 315 320

gcg ctg ctc tat atg ggc agc aag ctg acc acc aac gaa gag aca gtt 1008

Ala Leu Leu Tyr Met Gly Ser Lys Leu Thr Thr Asn Glu Glu Thr Val

325 330 335

gag ctc aac aag att ttc caa agg atg gac aaa aac gga gat ggc cag 1056

Glu Leu Asn Lys Ile Phe Gln Arg Met Asp Lys Asn Gly Asp Gly Gln

340 345 350

ctt gat aag cag gag ctc atg gaa ggc tac gtg gag ctg atg aag ctg 1104

Leu Asp Lys Gln Glu Leu Met Glu Gly Tyr Val Glu Leu Met Lys Leu

355 360 365

aaa ggc gaa gac gtt tcg gcg ctg gac cag agc gcc atc gag ttc gag 1152

Lys Gly Glu Asp Val Ser Ala Leu Asp Gln Ser Ala Ile Glu Phe Glu

370 375 380

gtg gag cag gtg ctc gac gct gtg gac ttt ggc aag aac ggc ttc ata 1200

Val Glu Gln Val Leu Asp Ala Val Asp Phe Gly Lys Asn Gly Phe Ile

385 390 395 400

gag tac tcg gag ttc gtc aca gtg gca atg gac cgc aag aca ctc ttg 1248

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

405 410 415

tct cgc cag cga ctg gaa aga gcc ttt gga atg ttc gac gct gac ggc 1296

Ser Arg Gln Arg Leu Glu Arg Ala Phe Gly Met Phe Asp Ala Asp Gly

420 425 430

tcg ggc aag att tcc tct tca gag ttg gcc acc ata ttc ggg gtg agc 1344

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

435 440 445

gag gtc gac tcc gaa acc tgg cgc cgc atc ctc gcg gag gtg gac aga 1392

Glu Val Asp Ser Glu Thr Trp Arg Arg Ile Leu Ala Glu Val Asp Arg

450 455 460

aac aac gac ggg gaa gtg gac ttt gag gag ttc cgg cag atg ctc ctg 1440

Asn Asn Asp Gly Glu Val Asp Phe Glu Glu Phe Arg Gln Met Leu Leu

465 470 475 480

aag ttg tgc gga gat acc gcg gcg gaattcccta ccctcgat atg tgc aaa 1491

Lys Leu Cys Gly Asp Thr Ala Ala Met Cys Lys

485 490

gta ctg atc ttt ggc tgt att tcg gta gca acg cta atg act aca gct 1539

Val Leu Ile Phe Gly Cys Ile Ser Val Ala Thr Leu Met Thr Thr Ala

495 500 505

tat gga gca tct cta tca tca gca aaa agg aaa cct ctt caa aca tta 1587

Tyr Gly Ala Ser Leu Ser Ser Ala Lys Arg Lys Pro Leu Gln Thr Leu

510 515 520

ata aag gat tta gaa ata ttg gaa aat atc aag aac aag att cat ctc 1635

Ile Lys Asp Leu Glu Ile Leu Glu Asn Ile Lys Asn Lys Ile His Leu

525 530 535

gag ctc tac aca cca act gag acc cag gag tgc acc cag caa act ctg 1683

Glu Leu Tyr Thr Pro Thr Glu Thr Gln Glu Cys Thr Gln Gln Thr Leu

540 545 550 555

cag tgt tac ctg gga gaa gtg gtt act ctg aag aaa gaa act gaa gat 1731

Gln Cys Tyr Leu Gly Glu Val Val Thr Leu Lys Lys Glu Thr Glu Asp

560 565 570

gac act gaa att aaa gaa gaa ttt gta act gct att caa aat atc gaa 1779

Asp Thr Glu Ile Lys Glu Glu Phe Val Thr Ala Ile Gln Asn Ile Glu

575 580 585

aag aac ctc aag agt ctt acg ggt cta aat cac acc gga agt gaa tgc 1827

Lys Asn Leu Lys Ser Leu Thr Gly Leu Asn His Thr Gly Ser Glu Cys

590 595 600

aag atc tgt gaa gct aac aac aag aaa aaa ttt cct gat ttt ctc cat 1875

Lys Ile Cys Glu Ala Ash Asn Lys Lys Lys Phe Pro Asp Phe Leu His

605 610 615

gaa ctg acc aac ttt gtg aga tat ctg caa 1905

Glu Leu Thr Asn Phe Val Arg Tyr Leu Gln

620 625

Claims (7)

1. an Eimeria tenella (Eimeria tenella) immunity regulating type DNA vaccine, this vaccine comprise and have wherein inserted the eucaryon plasmid carrier that Eimeria tenella calcium is transferred binding domain protein kinase gene pEtK2.

2. by the described dna vaccination of claim 1, wherein further also comprising links together with described gene pEtK2 inserts the chicken interleukin-2 gene of described eucaryon plasmid carrier.

3. by the described dna vaccination of claim 2, wherein further also be included in and insert one section protease Stuart factor between described gene pEtK2 and the gene chIL-2 _aSpecificity hydrolysis sequential coding nucleotide sequence GAA TTC CCT ACCCTC GAT forms a fusion gene pEtK2-X _a-chIL-2.

4. the described dna vaccination of claim 3, wherein said fusion gene has the nucleotide sequence shown in SEQ ID NO:1.

5. each described dna vaccination among the claim 1-4, wherein said eucaryon plasmid carrier is pcDNA3.0, pcDNA3.1, pVAX1.0 or pcDNA4/His Max.

6. each described dna vaccination in the claim claim 5, wherein said eucaryon plasmid carrier is pVAX1.0.

7. the purposes of each described dna vaccination in the pharmaceutical preparation of preparation prevention or treatment chicken coccidiosis among the claim 1-6.

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