阅读说明：本技术 一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗 (Carp and carp spring viremia oral vaccine capable of being added into feed ) 是由 林锋 陈建明 黄爱霞 郭建林 孙丽慧 姜建湖 于 2019-11-29 设计创作，主要内容包括：本发明公开了一种可添加于饲料中的鲤鱼鲤春病毒血症口服疫苗,通过芽孢表面展示技术将鲤春病毒血症病毒的G蛋白展示在芽孢表面,通过富集培养和芽孢诱导获得重组菌的芽孢,再经冷冻干燥制备出固态的重组疫苗。本发明可以通过添加到饲料中进行直接投喂,来避免或降低鲤鱼鲤春病毒病的暴发,对于鲤鱼鲤春病毒血症具有较好的免疫预防作用。(The invention discloses a carp spring viremia oral vaccine capable of being added into feed. The invention can avoid or reduce the outbreak of the spring viremia of the carp and the carp by directly feeding the feed, and has better immunoprophylaxis effect on the spring viremia of the carp and the carp.)

1. An oral carp and carp spring viremia vaccine capable of being added into feed is characterized by being prepared by the following steps: (1) screening for viral antigens

Selecting the G protein of spring viremia of carp virus as an antigen for constructing a nucleic acid vaccine;

(2) cloning and expression of G protein Gene

Cloning the amplified G protein gene into a pMD18-T vector, then transforming escherichia coli DH5 α to screen out a positive clone strain, identifying by double enzyme digestion of Nco I and Sac I, connecting to an intermediate bridge plasmid pET30a-G, carrying out double enzyme digestion by utilizing Sac I and Kpn I, cloning a target gene onto a plasmid pJS700 vector, and constructing an integrated recombinant plasmid pJS 700-G;

(3) bacillus subtilis surface display of G proteins

Transforming the integrative recombinant plasmid pJS700-G into competent cells to obtain a recombinant strain;

(4) cultivation of recombinant Bacillus

Culturing the recombinant strain in DSM culture medium, inducing to generate spore, freeze drying the spore, and storing at-80 deg.C.

2. The carp spring viraemia oral vaccine capable of being added into feed according to claim 1, wherein the use method comprises: in the feed processing process, the carp and carp spring viremia oral vaccine is directly added into feed raw materials and uniformly mixed to prepare feed with the vaccine for feeding.

3. The carp spring viraemia oral vaccine capable of being added into feed according to claim 1, wherein the use method comprises: before feeding, the carp and carp spring viremia oral vaccine is dissolved in water, sprayed on the feed, and fed after the feed is adsorbed and dried.

4. The oral carp spring viraemia vaccine according to claim 1, wherein the competent cell is Bacillus subtilis competent cell.

5. The oral carp and carp spring viraemia vaccine added to feed according to claim 1, wherein the primers for obtaining the G protein gene from the carp spring viraemia virus by PCR amplification are as follows:

a forward primer: 5'-CATGCCATGGATGTCTATCATCAGCTACATCGCA-3' the flow of the air in the air conditioner,

reverse primer: 5'-CGAGCTCAACGAAGGACCGCATTTCG-3' are provided.

Technical Field

The invention relates to the technical field of prevention and treatment of carp disease immunity, and particularly relates to a carp and carp spring viremia oral vaccine capable of being added into feed.

Background

Common carp is commonly called carpi raisin, carpi and the like, belongs to the family Cyprinus, is one of the main freshwater fish species in China, and accounts for more than half of the total amount of freshwater fish in China. Its body is flat and round, its mouth is horseshoe-shaped, and 2 pairs are needed. The back fin base is longer, and the back fin and the hip fin are both provided with a thick and strong hard spine with sawteeth. The body side is golden yellow, and the lower leaves of the tail fin are orange red. Common carps are usually inhabited on the bottom layer of water bodies of water plants in rivers, lakes, reservoirs and ponds, and mainly feed benthonic animals. Since carps contain rich nutrients, the carp feed has the effects of reducing cholesterol and preventing and treating arteriosclerosis and coronary heart disease, and is used as ornamental fish or edible fish in China and Japan, and is used as edible fish to be cultured in European countries such as Germany. Many varieties of artificially cultured carps, such as red carps, Cyprinus carpiod, koi, Cyprinus carpioi, Hibiscus carpio, and Cyprinus parvifolius, about 2900 varieties exist.

Diseases such as gill rot, scaly diseases, enteritis and the like frequently occur in the carp breeding process, and among them, Spring viremia of carp SVC (SVC for short) caused by Spring virus of carp is particularly serious. Spring viremia of carp, namely infectious ascites of carp, mainly infects carp, and has infectivity for silver carp, bighead carp, European crucian carp, grass carp and the like, which can cause morbidity and mortality, and is listed as epidemic disease to be declared by OIE. The virus only causes diseases when the temperature rises in spring, so the virus is called as spring carp virus disease, is an acute, hemorrhagic and infectious virus disease, is often epidemic in cyprinid fishes, particularly carps and koi, causes death of juvenile fishes and adult fishes, and is seriously harmful. The spring carp virus disease is a second type of infectious disease which is necessarily detected by international veterinary institute (OIE) and domestic in-and-out animals, so that the disease control work in breeding production needs to be well done. However, due to the relative lag of basic research work related to carp virus biology and the complexity of culture environment factors, the pathogenic mechanism of carp and carp spring viremia cannot be judged, so that the virus disease sometimes breaks out, and great economic loss is brought to farmers.

Disclosure of Invention

The invention aims to provide the carp and carp spring viremia oral vaccine capable of being added into feed, which can be directly fed by adding into the feed to avoid or reduce outbreak of carp and carp spring viremia and has a good immunoprophylaxis effect on the carp and carp spring viremia.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an oral vaccine of carp and carp spring viremia capable of being added into feed is prepared by the following steps:

(1) screening for viral antigens

Selecting the G protein of spring viremia of carp virus as an antigen for constructing a nucleic acid vaccine;

(2) cloning and expression of G protein Gene

Cloning the amplified G protein gene into a pMD18-T vector, then transforming escherichia coli DH5 α to screen out a positive clone strain, identifying by double enzyme digestion of Nco I and Sac I, connecting to an intermediate bridge plasmid pET30a-G, carrying out double enzyme digestion by utilizing Sac I and Kpn I, cloning a target gene onto a plasmid pJS700 vector, and constructing an integrated recombinant plasmid pJS 700-G;

(3) bacillus subtilis surface display of G proteins

Transforming the integrative recombinant plasmid pJS700-G into competent cells to obtain a recombinant strain;

(4) cultivation of recombinant Bacillus

Culturing the recombinant strain in DSM culture medium, inducing to generate spore, freeze drying the spore, and storing at-80 deg.C.

Spring viraemia of carp is an acute hemorrhagic infectious disease in the family carpidae caused by a rhabdovirus, Spring Viraemia of Carp Virus (SVCV), the SVCV genome is linear, single-stranded non-segmented negative-strand RNA, mainly comprises five open reading frames, and encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L). Through experimental exploration, the inventor selects SVCV G protein as antigen for constructing nucleic acid vaccine to obtain better effect.

Aiming at the problems that spring viremia of carp seriously harmed in the current carp industry is influenced by various factors, the incidence rate of the disease is high, the pathogenesis is complex, and no fish medicine with obvious curative effect is available in the market temporarily, so that vaccination and immunization are a better way. The commercial vaccines in the market are mainly inactivated vaccine and attenuated vaccine. The inactivated vaccine is also called as a killed vaccine, and is a vaccine prepared by killing complete pathogenic microorganisms cultured in large quantity by heating or physical and chemical methods such as formaldehyde and the like, so that the pathogens lose infectivity and toxicity and keep immunogenicity, and combining corresponding adjuvants. Because the inactivated vaccine needs to be used for a large amount of diseased fish tissues or cells, the defects of high cost, potential infection risk and the like exist, and the inactivated vaccine is difficult to popularize and apply in practice. According to the screened bacillus surface display carrier, the invention develops an oral vaccine which is low in cost and suitable for popularization and application, meets the epidemic prevention requirement of spring viremia of carps and carps in the culture process, and aims to reduce the morbidity and mortality of the carps in the culture process.

The using method of the carp and carp spring viremia oral vaccine comprises the following steps: in the feed processing process, the carp and carp spring viremia oral vaccine is directly added into feed raw materials and uniformly mixed to prepare feed with the vaccine for feeding.

The using method of the carp and carp spring viremia oral vaccine comprises the following steps: before feeding, the carp and carp spring viremia oral vaccine is dissolved in water, sprayed on the feed, and fed after the feed is adsorbed and dried.

The competent cell is Bacillus subtilis competent cell.

The primers adopted for amplifying and obtaining the G protein gene from the spring viremia of carp virus by the PCR method are as follows:

a forward primer: 5'-CATGCCATGGATGTCTATCATCAGCTACATCGCA-3' the flow of the air in the air conditioner,

reverse primer: 5'-CGAGCTCAACGAAGGACCGCATTTCG-3' are provided.

The invention has the beneficial effects that: according to the characteristics of high temperature resistance and water quality adjustment of the bacillus subtilis spore, the G protein of the spring viremia of carp virus is displayed on the surface of the spore by a spore surface display technology. The spores of the recombinant bacteria are obtained through enrichment culture and spore induction, the solid recombinant vaccine is prepared through freeze drying, and the recombinant vaccine with different concentrations is added into the feed according to the immune protection requirement, so that the defects that the existing oral vaccine cannot resist high temperature and cannot be added into the feed are overcome, the absorption of the carp to a target antigen is facilitated, and the method is suitable for screening and preventing spring viremia of the carp and the carp.

Drawings

FIG. 1 is a diagram of the construction of an integrative plasmid.

FIG. 2 is a carp spring virus G protein recombinant protein expression diagram. M: protein Marker, 48KDa and 63KDa are the sizes of the strip proteins, and 58.59KDa is the target protein; 1: coli.BL21(DE3) pET-30a empty plasmid; 2: not inducing; 3-7: IPTG was added to the cells to induce final concentrations of 1.0, 0.8, 0.6, 0.4, 0.2mM, respectively.

FIG. 3 is a colony counting chart of the recombinant bacillus spore freeze-dried powder. The upper row of plates is diluted by freeze-dried powder 10¹¹3 replicates of the previous panel, the next panel was 10 dilutions of lyophilized powder¹⁰Replicate 3 times, spore concentration averaged.

FIG. 4 is a graph showing the analysis of the concentration of recombinant Bacillus species pre-mixed with a feed product and then sampled and cultured. Blank group: feed without any bacillus added; wild type: addition 10⁹cpu/ml feed of wild type bacillus; test 1: addition 10⁹cpu/ml feed of recombinant bacillus; test 2: addition 10¹⁰cpu/ml feed of recombinant bacillus; test 3: addition 10¹¹cpu/ml feed of recombinant bacillus.

FIG. 5 serum antibody levels TAS-ELISA assay at different time intervals after oral vaccine immunization. 1. PSB control; 2. oral immunization group 1: continuous immunization for 14 days; 3. oral immunization group 2: the feeding is stopped for 3 days after 7 days of continuous feeding, and the process is repeated for 3 times.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

Material

Spring Viremia of Carp Virus (SVCV) is provided by fish disease research institute of freshwater aquaculture research in Zhejiang province; normal carps were purchased from a certain farm in Jinhua city, Zhejiang province; the bacillus and the expression vector are provided by university academy of college of university in Jiangsu; various enzymes, PCR reagents, and media were purchased from Takara; primer synthesis, other consumables were purchased from Shanghai Biometrics.