Composite recombinant plasmid based on Prime-boost immunity and chicken coccidiosis vaccine

Abstract

The invention discloses a composite recombinant plasmid based on Prime-boost immunity and a chicken coccidiosis vaccine. The composite recombinant plasmid comprises a plurality of single recombinant plasmids which are correspondingly obtained by respectively recombining eimeria tenella 14-3-3 genes onto a plurality of different expression vectors; wherein the plurality of single recombinant plasmids at least comprise a recombinant eukaryotic plasmid and a recombinant prokaryotic plasmid. Aiming at the problems that in the prior art, chicken coccidiosis mainly depends on drugs and live oocyst vaccines, but anti-coccidiosis drugs have drug resistance, and the live oocyst vaccines have toxicity and intestinal injury, the invention provides a heterogeneity immunity enhancing method based on the same antigen, while the problems existing in the existing vaccines are solved, the immunogenicity of the chicken is improved, and the immunogenicity of the chicken is improved. The invention further provides a composite recombinant plasmid and a chicken coccidiosis vaccine capable of improving the epidemic prevention effect of the vaccine.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a composite recombinant plasmid, a recombinant protein and a chicken coccidiosis vaccine based on Prime-boost immunity. Background Art

[0002] Chicken coccidiosis is a parasitic protozoan disease that seriously affects the development of the global poultry industry. It can damage the host intestine, reduce feed conversion rate, increase host mortality and susceptibility to other pathogens. The economic losses caused by chicken coccidiosis are as high as 10.4 billion pounds per year worldwide. There are seven internationally recognized Eimeria species that infect chickens. In clinical practice, chicken coccidiosis is usually caused by mixed infection of these Eimeria species. Therefore, a single vaccine is often unable to resist naturally occurring coccidiosis. On this basis, the development of vaccines that can effectively prevent mixed infection of multiple Eimeria species has become a research focus in this field.

[0003] At present, the prevention and control of chicken coccidiosis mainly relies on drugs and live oocyst vaccines, but there is no effective solution to the problems of drug resistance of anticoccidial drugs, toxicity of live oocyst vaccines, and intestinal damage. The new genetically engineered vaccine has high safety and low production cost. However, at this stage, the implementation of genetically engineered vaccines for chicken coccidiosis mainly adopts the homogeneous Prime-boost immunization strategy. Traditional homogeneous Prime-boost immunization is to strengthen the same vaccine, which can only improve the body's humoral immunity level and has little effect on the cellular immunity level.

[0004] Therefore, in view of the drawbacks of the above-mentioned prior art, the present invention provides a heterogeneous booster immunization method based on the same antigen on the basis of comprehensive consideration of the booster immunization strategy, which not only provides candidate antigens for the development of vaccines for the prevention and control of chicken coccidiosis, but also provides ideas for the optimization of the immunization program of chicken coccidiosis genetic engineering vaccines. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a heterogeneous booster immunization method based on the same antigen, which can overcome the problems of the existing vaccines and further provide a composite recombinant plasmid and chicken coccidiosis vaccine based on Prime-boost immunity to improve the vaccine's preventive effect.

[0006] In order to achieve the above object, the present invention provides a composite recombinant plasmid based on Prime-boost immunity, wherein the composite recombinant plasmid comprises a plurality of single recombinant plasmids correspondingly obtained by respectively recombining the Eimeria tenella 14-3-3 gene into a plurality of different expression vectors; wherein,

[0007] The plurality of single recombinant plasmids at least include recombinant eukaryotic plasmids and recombinant prokaryotic plasmids.

[0008] Preferably, the expression vector used to construct the recombinant eukaryotic plasmid is selected from the pVAX1.0 plasmid, and the expression vector used to construct the recombinant prokaryotic plasmid is selected from the pET-32a plasmid.

[0009] The present invention also provides a chicken coccidiosis vaccine based on Prime-boost immunity, wherein the chicken coccidiosis vaccine comprises the composite recombinant plasmid as described above; or,

[0010] The chicken coccidiosis vaccine comprises the composite recombinant protein obtained by inducing expression of the composite recombinant plasmid as described above; or,

[0011] The chicken coccidiosis vaccine comprises a portion of the single recombinant plasmid in the composite recombinant plasmid as described above, and a recombinant protein induced to be expressed by another portion of the single recombinant plasmid.

[0012] Here, the two simplest single recombinant plasmids are used for description. For example, the composite recombinant plasmid here is composed of a recombinant eukaryotic plasmid and a recombinant prokaryotic plasmid. Then, the chicken coccidiosis vaccine here has four composition forms: 1. The chicken coccidiosis vaccine is composed of this recombinant eukaryotic plasmid and this recombinant prokaryotic plasmid; 2. The chicken coccidiosis vaccine is composed of the recombinant proteins induced to express by this recombinant eukaryotic plasmid and this recombinant prokaryotic plasmid, that is, the vaccine includes the first recombinant protein induced to express by the recombinant eukaryotic plasmid and the second recombinant protein induced to express by the recombinant prokaryotic plasmid; 3. The chicken coccidiosis vaccine is composed of the recombinant protein induced to express by the recombinant eukaryotic plasmid, and the recombinant prokaryotic plasmid; 4. The chicken coccidiosis vaccine is composed of the recombinant protein induced to express by the recombinant prokaryotic plasmid, and the recombinant eukaryotic plasmid. The third and fourth vaccines each include a recombinant plasmid and a recombinant protein. Preferably, the chicken coccidiosis vaccine includes a recombinant eukaryotic plasmid, and a recombinant protein induced to express by the recombinant prokaryotic plasmid. That is, the fourth situation is a preferred embodiment of the present invention. It should be noted that although the fourth scheme is preferred here, the above three schemes can also be constructed to form a chicken coccidiosis vaccine based on the heterogeneous Prime-boost immune strategy. Therefore, the present invention is not limited to scheme 4, and the other three schemes can also be selected and adopted in a targeted manner according to actual conditions. Of course, it is not limited to the scheme with only one recombinant prokaryotic plasmid and one recombinant eukaryotic plasmid. The types of recombinant prokaryotic plasmids and recombinant eukaryotic plasmids can select corresponding prokaryotic vectors and eukaryotic vectors according to actual conditions, and correspondingly construct a variety of recombinant prokaryotic plasmids and recombinant eukaryotic plasmids, which will not be elaborated here.

[0013] The preparation of each recombinant protein can refer to the inducible expression method that can be understood by those skilled in the art. For the differences between eukaryotic plasmids and prokaryotic plasmids in induced expression, those skilled in the art can make adjustments according to the situation within the scope that can be understood by those skilled in the art.

[0014] Of course, in a more preferred embodiment of the present invention, the preparation method of each recombinant protein specifically includes:

[0015] S100, extracting total RNA of Eimeria tenella and reverse transcription to obtain cDNA;

[0016] S200, amplifying the cDNA obtained in step S100 using amplification primers to obtain an amplified product;

[0017] S300, connecting the obtained amplified product with the intermediate vector and culturing the resultant to obtain a transitional recombinant plasmid;

[0018] S400, using a set of restriction endonucleases to double-digest the transitional recombinant plasmid and expression vector, recovering the target gene fragment, inducing expression, purifying, and obtaining the recombinant protein.

[0019] Preferably, the base length of the amplified product in step S200 is 846 bp.

[0020] Preferably, when the expression vector is a eukaryotic expression vector, the amplification primers in step S200 are first amplification primers as shown in SEQ ID No: 1 and SEQ ID No: 2;

[0021] When the expression vector is a prokaryotic expression vector, the amplification primers in step S200 are second amplification primers as shown in SEQ ID No: 3 and SEQ ID No: 4;

[0022] Preferably, when the expression vector is a eukaryotic expression vector, the restriction endonuclease in step S400 is selected from HindⅢ and XhoⅠ;

[0023] When the expression vector is a prokaryotic expression vector, the restriction endonuclease in step S400 is selected from BamH I and HindIII.

[0024] Preferably, in step S300, the intermediate vector is selected from pMD-18T vector.

[0025] Through the above technical scheme, the present invention is based on heterogeneous Prime-boost immunity, takes the Et14-3-3 gene as the target gene, and constructs a chicken coccidiosis vaccine based on the synergy of recombinant eukaryotic plasmid and recombinant prokaryotic plasmid. Compared with the prior art, its immune performance against chicken coccidiosis vaccine is greatly enhanced, providing a new idea for the immune optimization of chicken coccidiosis vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0027] Figure 1 It is a double enzyme digestion identification result diagram of the recombinant eukaryotic plasmid pVAX-Et14-3-3 in Verification Example 2; wherein, lane M is a molecular weight marker, lane 1 is pVAX-Et14-3-3 after double enzyme digestion with HindⅢ and XhoⅠ, and lane 2 is pVAX-Et14-3-3 without enzyme digestion;

[0028] Figure 2 It is a double enzyme digestion identification result diagram of the recombinant prokaryotic plasmid pET-32a-Et14-3-3 in Verification Example 2; wherein, lane M is a molecular weight marker, lane 1 is pET-32a-Et14-3-3 after double digestion with BamH I and Hind III, and lane 2 is pET-32a-Et14-3-3 without enzyme digestion;

[0029] Figure 3 : is the SDS-PAGE electrophoresis analysis result of the purified rEt14-3-3 recombinant protein in Verification Example 3; wherein, lane M is a molecular weight marker, lane 1 is a 0.5 mg / mL BSA control, and lane 2 is the purified rEt14-3-3 recombinant protein;

[0030] Figure 4 It is a Western blot analysis result diagram of the purified rEt14-3-3 recombinant protein in Verification Example 3; wherein, lane M is a molecular weight marker, and lane 1 is the purified rEt14-3-3 recombinant protein. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0032] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0033] The chickens used in the present invention were purchased from 320 one-day-old sea blue white male chicks (weighing about 40 g) provided by a chicken farm in Jiangsu Province. The chickens were raised in a disinfected and coccidia-free environment with free access to food and water. The Eimeria tenella (E. tenella) oocysts were provided by the Veterinary Parasitology Teaching and Research Section of Nanjing Agricultural University.

[0034] The pVAX1.0 eukaryotic plasmid and the pET-32a prokaryotic plasmid are conventional commercial products of Invitrogen; the plasmid mini kit (brand number D6943-01) is a conventional commercial product of OMEGA; the reverse transcription kit (brand number MR201-01) and DH5α competent cells are conventional commercial products of Vazyme; the RNA extraction kit (brand number 9108) and the restriction endonucleases HindⅢ, XhoⅠ and BamH I are conventional commercial products of TaKaRa; the mouse anti-His tag monoclonal antibody (1:5000 dilution) and the HRP-labeled goat anti-mouse IgG (1:10000 dilution) are conventional commercial products of Solebo.

[0035] Preparation Example 1: Construction of composite recombinant plasmid

[0036] Primer design: Primers were designed based on the coding region sequence of the Et14-3-3 gene (i.e., the Eimeria tenella 14-3-3 gene, whose GenBank accession number is XM_013374079.1), and the base length of the amplified product was estimated to be about 846 bp. The primer sequences and restriction sites are specifically shown in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] RNA extraction and reverse transcription: 3 mL of purified E. tenella sporulated oocysts were taken to extract total RNA, which was then reverse transcribed into the first-strand cDNA (single-stranded cDNA). The single-stranded cDNA was used as a PCR template and amplified using the two sets of primer pairs shown in Table 1. The obtained amplification products (two sets of primer pairs were used here to amplify two amplification products) were respectively recovered by gel, i.e., the two amplification products were recovered by gel separately.

[0041] Ligation and transformation: The two amplification products recovered from the gel were connected to the pMD-18T vector respectively, and then transferred into DH5α competent cells and cultured at 37°C for 12-14 hours. The monoclonal clones after culture were identified by restriction digestion, and after successful identification, they were sent to Nanjing GenScript for sequencing. The sequencing results were analyzed by DNAStar and compared with the Et14-3-3 gene sequence to obtain the first transitional recombinant plasmid pMD-18T-Et14-3-3-1 and the second transitional recombinant plasmid pMD-18T-Et14-3-3-2.

[0042] Construction of composite recombinant plasmid: The first transitional recombinant plasmid pMD-18T-Et14-3-3-1 and the eukaryotic expression vector pVAX1.0 were double-digested with HindⅢ and XhoⅠ, and the second transitional recombinant plasmid pMD-18T-Et14-3-3-2 and the prokaryotic expression vector pET-32a were double-digested with BamH I and Hind III, and the target gene fragment was recovered, connected, transformed, and identified by enzyme digestion. The recombinant eukaryotic plasmid pVAX-Et14-3-3 and the recombinant prokaryotic plasmid pET-32a-Et14-3-3 were obtained, and the two were mixed to form a composite recombinant plasmid.

[0043] Preparation Example 2: Preparation of recombinant protein based on recombinant prokaryotic plasmid pET-32a-Et14-3-3

[0044] The recombinant prokaryotic plasmid pET-32a-Et14-3-3 identified in Preparation Example 1 was transformed into E.Coli BL21 competent cells for culture to obtain a bacterial solution containing the recombinant prokaryotic plasmid pET-32a-Et14-3-3. The bacterial solution containing the recombinant prokaryotic plasmid pET-32a-Et14-3-3 was inoculated into LB medium (Ampicillin resistance, 100g / mL), cultured at 37°C, 180rpm until the OD value was 0.6, and IPTG at a concentration of 1mmol / L was added, induced at 37°C for 5h, and centrifuged after 4 ultrasonications (centrifugation conditions were 13000rpm, 8min), to obtain a recombinant protein based on the recombinant prokaryotic plasmid pET-32a-Et14-3-3 (referred to as rEt14-3-3). A small amount of the precipitate and supernatant of rEt14-3-3 were taken for SDS-PAGE electrophoresis analysis.

[0045] Verification Example 1: Sequencing results of amplified products

[0046] The amplified product after amplification in Preparation Example 1 was sequenced, and the sequencing results showed that the Et14-3-3 coding region fragment was successfully amplified, the length of the amplified target fragment was consistent with the expected size (846 bp) and the sequencing was correct.

[0047] Verification Example 2: Identification of recombinant eukaryotic plasmids and recombinant prokaryotic plasmids

[0048] The recombinant eukaryotic plasmid pVAX-Et14-3-3 and the recombinant prokaryotic plasmid pET-32a-Et14-3-3 prepared in Preparation Example 1 were identified after double enzyme digestion. The identification results are as follows: Figure 1 and Figure 2 As shown, Figure 1 This is the result of double enzyme digestion identification of the recombinant eukaryotic plasmid pVAX-Et14-3-3. Figure 2 The figure shows the double enzyme digestion result of the recombinant prokaryotic plasmid pET-32a-Et14-3-3. Figure 1 and Figure 2 It can be seen that after double enzyme digestion, the recombinant eukaryotic plasmid and the recombinant prokaryotic plasmid constructed by the present invention both showed a target band of about 840 bp in size, which was consistent with expectations, and the sequencing results were correct.

[0049] Verification Example 3: Identification of Recombinant Protein

[0050] The results of SDS-PAGE electrophoresis analysis of the purified rEt14-3-3 recombinant protein in Preparation Example 2 are as follows: Figure 3 As shown. Figure 3 It can be seen that the rEt14-3-3 prepared in Preparation Example 2 has a high purity and a size of about 49.7 KD, which is consistent with expectations.

[0051] After SDS-PAGE protein electrophoresis, rEt14-3-3 in Preparation Example 2 was transferred to a PVDF membrane. It was then blocked in 5% BSA at 37°C for 2 hours. Western blot analysis of the reactivity of Et14-3-3: the primary antibody was mouse anti-His tag monoclonal antibody, incubated at 37°C for 2 hours, and the secondary antibody was HRP-labeled goat anti-mouse IgG, incubated at 37°C in the dark for 2 hours. The results of the Western blot analysis are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that a single reaction band appeared in the Western blot analysis of rEt14-3-3, with a size of approximately 49.7KD, which was consistent with expectations.

[0052] The above identification proves that the present invention produces the expected recombinant protein.

[0053] Application Examples

[0054] A total of 240 14-day-old chickens of similar weight and good condition were selected. The corresponding reagents were injected into the leg muscles at 14 and 21 days of age, respectively, and PBS reagents or Eimeria tenella oocysts were orally inoculated at 28 days of age. The specific immunization experimental groups and the corresponding injection and inoculation reagents at the corresponding age are shown in Table 2.

[0055] Specifically, PBS200μL·feather -1 Each chicken was injected with 200 μL of PBS buffer solution; 5×10 oocysts 4 Feather -1 Refers to oral inoculation of 5×10 4 Eimeria tenella oocysts; pVAX1.0 empty plasmid 100 μg· -1 Refers to each chicken being injected with 100 μg of pVAX1.0 eukaryotic plasmid and 200 μg of pET-32a protein. -1Refers to the injection of 200μg of pET-32a prokaryotic plasmid directly expressed protein per chicken; pVAX-Et14-3-3 plasmid 100μg per chicken -1 Each chicken was injected with 100 μg of the recombinant eukaryotic plasmid pVAX-Et14-3-3 prepared in Preparation Example 1; 200 μg of pET-32a-Et14-3-3 protein per chicken. -1 This means that each chicken is injected with 200 μg of the recombinant protein rEt14-3-3 prepared in Preparation Example 2.

[0056] Table 2

[0057]

[0058]

[0059] On the 7th day after the attack, all chickens were slaughtered and the protection effect was evaluated by calculating the anti-coccidial index. The main collected indicators were average weight gain, oocyst excretion, lesion score, etc.

[0060] Test Example 1

[0061] The cecum and oocyst output of the multiple groups of experimental chickens in the corresponding examples were tested respectively, and the results of the cecum lesion scores and ovarian output are shown in Table 3.

[0062] Table 3

[0063]

[0064]

[0065] It can be seen from Table 3 that the egg cyst discharge volume and lesion score of Et14-3-3 homogeneous immunization test group 1, Et14-3-3 homogeneous immunization test group 2, Et14-3-3 heterogeneous immunization test group 1, and Et14-3-3 heterogeneous immunization test group 2 were significantly lower than those of the non-immune non-infected control group (P<0.05), indicating that both Et14-3-3 homogeneous immunization and heterogeneous immunization can resist the invasion of E. tellena, reduce egg cyst production, and alleviate chicken cecal damage (P<0.05). Among them, the egg cyst discharge volume of the Et14-3-3 heterogeneous immunization test group was significantly lower than that of the Et14-3-3 homogeneous immunization test group (P<0.05), indicating that the vaccine based on heterogeneous Prime-boost immunization provided by the present invention can further reduce egg cyst production.

[0066] Test Example 2

[0067] Corresponding to the multiple groups of experimental chickens in the use case, the parameters such as the chicken anticoccidial index after immunization were tested respectively (see Table 4 for details), and the results obtained are shown in Table 4.

[0068] Table 4

[0069]

[0070]

[0071] It can be seen from Table 4 that compared with the non-immune non-infected control group, the average weight gain of Et14-3-3 homogeneous immunization test group 1, Et14-3-3 homogeneous immunization test group 2, Et14-3-3 heterogeneous immunization test group 1, and Et14-3-3 heterogeneous immunization test group 2 was significantly increased (P<0.05), and the average weight gain of Et14-3-3 heterogeneous immunization test groups 1 and 2 was significantly higher than that of Et14-3-3 homogeneous immunization test groups 1 and 2 (P<0.05), indicating that the Et14-3-3 heterogeneous immunization test group significantly increased the weight of chickens. The anti-coccidial index of Et14-3-3 homogeneous immunization and heterogeneous immunization after infection with E. tellena was greater than 160. This shows that both Et14-3-3 homogeneous immunity and heterogeneous immunity can provide a certain degree of protection against the invasion of E. tellena. The anti-coccidial index of the Et14-3-3 heterogeneous immunization test group was greater than that of the homogeneous immunization test group, indicating that the heterogeneous Prime-boost immunization strategy provided by the present invention has a good effect.

[0072] The technical solution of the present invention is based on heterogeneous Prime-boost immunity, and through the cloning and expression of the common antigen 14-3-3, a 14-3-3 DNA vaccine and a recombinant subunit vaccine are constructed. In the actual chicken coccidiosis virus immunization, compared with the traditional homogeneous immunization strategy, the anti-coccidial index is effectively improved, providing an optimized vaccine for the prevention and control of chicken coccidiosis.

[0073] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0074] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0075] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A composite recombinant plasmid based on Prime-boost immunity, characterized in that: The composite recombinant plasmid includes a plurality of single recombinant plasmids obtained by respectively recombining the Eimeria tenella 14-3-3 gene into a plurality of different expression vectors; wherein, The plurality of single recombinant plasmids at least include recombinant eukaryotic plasmids and recombinant prokaryotic plasmids.

2. The composite recombinant plasmid according to claim 1, characterized in that: The expression vector used to construct the recombinant eukaryotic plasmid is selected from the pVAX1.0 plasmid, and the expression vector used to construct the recombinant prokaryotic plasmid is selected from the pET-32a plasmid.

3. A chicken coccidiosis vaccine based on Prime-boost immunity, characterized in that: The chicken coccidiosis vaccine comprises the composite recombinant plasmid as claimed in claim 1 or 2; or The chicken coccidiosis vaccine comprises the composite recombinant protein obtained by inducing expression of the composite recombinant plasmid as claimed in claim 1 or 2; or The chicken coccidiosis vaccine comprises a portion of a single recombinant plasmid in the composite recombinant plasmid as claimed in claim 1 or 2, and a recombinant protein induced to be expressed by another portion of the single recombinant plasmid.

4. The chicken coccidia vaccine according to claim 3, characterized in that The chicken coccidia vaccine comprises a recombinant eukaryotic plasmid and a recombinant protein induced to be expressed by the recombinant prokaryotic plasmid.

5. The chicken coccidia vaccine according to claim 3 or 4, characterized in that The preparation method of each recombinant protein includes: S100, extracting total RNA of Eimeria tenella and reverse transcription to obtain cDNA; S200, amplifying the cDNA obtained in step S100 using amplification primers to obtain an amplified product; S300, connecting the obtained amplified product with the intermediate vector and culturing the resultant to obtain a transitional recombinant plasmid; S400, using a set of restriction endonucleases to double-digest the transitional recombinant plasmid and expression vector, recovering the target gene fragment, inducing expression, purifying, and obtaining the recombinant protein.

6. The chicken coccidia vaccine according to claim 5, characterized in that The base length of the amplified product in step S200 is 846 bp.

7. The chicken coccidia vaccine according to claim 5, characterized in that When the expression vector is a eukaryotic expression vector, the amplification primers in step S200 are first amplification primers as shown in SEQ ID No: 1 and SEQ ID No: 2; When the expression vector is a prokaryotic expression vector, the amplification primers in step S200 are second amplification primers as shown in SEQ ID No: 3 and SEQ ID No:

4.

8. The chicken coccidia vaccine according to claim 5, characterized in that When the expression vector is a eukaryotic expression vector, the restriction endonuclease in step S400 is selected from HindⅢ and XhoⅠ; When the expression vector is a prokaryotic expression vector, the restriction endonuclease in step S400 is selected from BamH I and Hind III.

9. The chicken coccidia vaccine according to claim 5, characterized in that In step S300, the intermediate vector is selected from the pMD-18T vector.

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