CN102266556A - P32 gene based DNA (deoxyribonucleic acid) vaccine for goat pox, and preparation method thereof - Google Patents

Abstract

The invention discloses a P32 gene based DNA (deoxyribonucleic acid) vaccine for goat pox, and a preparation method thereof. The P32 gene based DNA vaccine contains the eukaryotic expression plasmid pVAX1-P32 of the P32 gene of the goat pox virus, and the plasmid concentration is 3mg-10mg/mL. The oral vaccine for goat pox disclosed by the invention is safe and effective, and can induce a goat body to generate specific humoral immunity, cellular immunity and mucosal immune response, thus effectively preventing the occurrence and prevalence of goat pox.

Description

A kind of based on P32 gene goatpox dna vaccination and preparation method

Technical field

The present invention relates to a kind of based on P32 gene goatpox dna vaccination and preparation method.

Background technology

Goatpox is a kind of acute, hot, the height contagious disease that is caused goat and sheep by the Capripoxvirus goat capripoxvirus, has higher M ﹠ M, is International Office of Epizootics (OIE) category-A animal epidemic and China's one class animal epidemic.Goatpox is generally popular in China, has almost spread all over all sheep countries, has caused great loss for the development of sheep husbandry, and particularly the goat aquaculture that is surging forward in the west area has been brought severe threat.

At present, it is main that China mainly takes vaccination to the control of goatpox, no matter but be inactivated vaccine or attenuated vaccine, all there is certain deficiency, not good as the inactivated vaccine immune effect, need be bigger with dosage, of short duration protection can only be provided; The attenuated vaccine immunity effect is better than inactivated vaccine, but can stimulate the varioliform exanthema reaction after the inoculation, also causes secondary infection when serious, and has virulence to return strong risk.And dna vaccination claims naked vaccine, it is the third generation vaccine after inactivated vaccine, attenuated live vaccine and genetic engineering recombinant protein vaccine, be used at present the prevention and control of multiple pathogenic infection diseases such as HIV, influenza virus, Plasmodium falciparum, and obtained positive progress.

Data shows according to the study, the P32 albumen of goat capripoxvirus P32 coded by said gene is the present structural protein of the total and high specificity of the goat capripoxvirus of all isolation identification all over the world, has stronger antigenicity, can induce the reaction of goat cellular immunization and humoral immunoresponse(HI), produce specificity neutralizing antibody and various cytokine, in the prevention of goatpox and control, play a significant role.

Do not appear in the newspapers about goatpox dna vaccination based on the P32 gene.

Summary of the invention

The technical problem to be solved in the present invention is, provide a kind of based on P32 goatpox dna vaccination and preparation method, this vaccine can induce goat to produce good humoral immunity and cellullar immunologic response, and does not cause that goat produces untoward reaction, has good biological safety.

Technical scheme of the present invention: contain the eukaryon expression plasmid pVAX1-P32 of goat capripoxvirus P32 gene, plasmid concentration is 3mg ~ 10mg/mL.

Its preparation method may further comprise the steps:

The first, adopt PCR method from the pMD-18T-P32 plasmid, to amplify the P32 gene, use H IndIII+B AmThe H I is carried out enzyme action, handles carrier for expression of eukaryon pVAX1 with method, then electrophoretic separation and reclaim genes of interest respectively;

The second, adopt T ₄Dna ligase inserts the P32 gene among the eukaryon expression plasmid pVAX1, transformed into escherichia coli DH5a, blue white macula screening, the suspicious bacterium colony of picking is cultivated 12 ~ 18h in the LB liquid culture based on 36 ℃ ~ 38 ℃, collect bacterium liquid and adopt the DNA extraction test kit to extract plasmid, identify with PCR and double digestion;

The 3rd, get and contain the recombination bacillus coli DH5a that is accredited as positive recombiant plasmid, after 36 ℃ ~ 38 ℃ of LB fluid mediums are cultivated 12 ~ 18h, adopt plasmid extraction kit to extract and obtain pVAX1-P32, be based on P32 gene goatpox dna vaccination.

Beneficial effect of the present invention: the present invention is applicable to the immunity inoculation of goatpox, and the generation of anti-system goatpox and popular has good economic benefit and ecological benefits simultaneously.The present invention compared with prior art has following technical advantage and good effect:

(1) immune effect is good: the goatpox dna vaccination that the present invention adopts carrier for expression of eukaryon pVAX1 to make up, and can induce goat to produce good humoral immunity and cellullar immunologic response behind the intramuscular injection animal;

(2) preparation cost is lower: dna vaccination of the present invention after the bacillus coli DH 5 alpha that only needs to contain recombiant plasmid pVAX1-P32 is cultivated in a large number, extracts recombiant plasmid pVAX1-P32 and gets final product when needed;

(3) immunity inoculation is convenient: dna vaccination of the present invention adopts intramuscular injection to inoculate, and is easy to operate, requires Intradermal or subcutaneous injection unlike the goatpox attenuated vaccine;

(4) safety has no side effect: dna vaccination of the present invention can not cause that virulence strengthens or causes gene integration, can not cause that goat edema, suppuration or general takes place send out symptoms such as pox yet.

Description of drawings

Fig. 1 is the preparation flow figure of goatpox dna vaccination of the present invention.

The specific embodiment

Embodiments of the invention: the primary raw material of goatpox dna vaccination of the present invention is to be made of the cloned plasmids pMD-18T-P32 that contains goat capripoxvirus P32 gene, carrier for expression of eukaryon pVAX1 and escherichia coli DH5a.

As schematically shown in Figure 1, the preparation method of goatpox dna vaccination of the present invention may further comprise the steps:

The first, the obtaining of goat capripoxvirus P32 gene: adopt to contain H IndIII and B AmThe goat capripoxvirus P32 gene-specific primer of H I restriction enzyme site amplifies goat capripoxvirus P32 gene from cloned plasmids pMD-18T-P32, agarose reclaims test kit and reclaims purification;

The second, the structure of recombinant eukaryon expression vector pVAX1-P32: adopt T ₄The goat capripoxvirus P32 gene that dna ligase obtains step 1, the eukaryotic expression matter carrier pVAX1 that handles with same enzyme action is connected, transformed into escherichia coli DH5a competent cell, through blue white macula screening, the suspicious bacterium colony of picking is cultivated 12h in the LB liquid culture based on 37 ℃, collects bacterium liquid and adopts DNA extraction test kit extraction plasmid to carry out PCR and double digestion evaluation;

The 3rd, a large amount of extractions of recombinant eukaryon expression vector pVAX1-P32: get the escherichia coli DH5a that contains positive recombinant eukaryon expression vector pVAX1-P32, after 37 ℃ of LB fluid mediums are cultivated 12h, centrifugal collection bacterium liquid adopts plasmid extraction kit, extract recombinant eukaryon expression vector pVAX1-P32, adjusting its concentration is 4.85mg/mL, is based on P32 gene goatpox dna vaccination.

Claims (2)

1. one kind based on P32 gene goatpox dna vaccination, and it is characterized in that: contain the eukaryon expression plasmid pVAX1-P32 of goat capripoxvirus P32 gene, plasmid concentration is 3mg ~ 10mg/mL.

2. a kind of preparation method as claimed in claim 1 based on P32 gene goatpox dna vaccination, it is characterized in that: it may further comprise the steps:

The first, adopt PCR method from the pMD-18T-P32 plasmid, to amplify the P32 gene, use H IndIII+B AmThe H I is carried out enzyme action, handles carrier for expression of eukaryon pVAX1 with method, then electrophoretic separation and reclaim genes of interest respectively;

The second, adopt T ₄Dna ligase inserts the P32 gene among the eukaryon expression plasmid pVAX1, transformed into escherichia coli DH5a, blue white macula screening, the suspicious bacterium colony of picking is cultivated 12 ~ 18h in the LB liquid culture based on 36 ℃ ~ 38 ℃, collect bacterium liquid and adopt the DNA extraction test kit to extract plasmid, identify with PCR and double digestion;

The 3rd, get and contain the recombination bacillus coli DH5a that is accredited as positive recombiant plasmid, after 36 ℃ ~ 38 ℃ of LB fluid mediums are cultivated 12 ~ 18h, adopt plasmid extraction kit to extract and obtain pVAX1-P32, be based on P32 gene goatpox dna vaccination.

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