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Intersecting protective vaccine antigen, preparation method and application thereof

A vaccine antigen and protective technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as the inability to meet industrial requirements, and achieve the effects of high recovery rate, high recovery rate and simple recovery method

Inactive Publication Date: 2009-09-02
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, due to the diversity and variability of biological pathogens in aquaculture, specific vaccines against a single pathogen can no longer meet industry requirements, while cross-vaccines that have protective effects against a variety of different pathogens are preferred due to their greater practical application value. Become the trend of vaccine development in the future

Method used

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  • Intersecting protective vaccine antigen, preparation method and application thereof
  • Intersecting protective vaccine antigen, preparation method and application thereof
  • Intersecting protective vaccine antigen, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The cross-protective vaccine antigen of the present invention has the vhdQ base sequence in the sequence table SEQ ID No.1 (see figure 2 ).

[0029] Antigen preparation method:

[0030] 1) Construction of plasmid pET258

[0031] Plasmid pET25 (purchased from Novogen, USA) was digested with BglII / NdeI to recover a 104bp fragment; plasmid pET28 (purchased from Novogen, USA) was digested with BglII / NdeI to recover a 5.3kb fragment; The DNA fragments were ligated with T4 DNA ligase, and the ligated solution was transformed into E. coli DH5α by the Hanahan method, and cultured on LB solid medium containing kanamycin (Kn, 50ug / ml) for 24 hours, and then screened for resistance to kanamycin Transformants, pick 2 transformants, extract plasmids, and verify that they are correctly recombined plasmids by sequencing. This plasmid was named pET258.

[0032] 2) Construction of plasmid pEVQ:

[0033] a) Using Vibrio harveyi T4 as a template, use the following primers to PCR vhd...

Embodiment 2

[0069]The above-mentioned obtained cross-protective vaccine antigen VhdQ is purified by the following method: the centrifuged supernatant lysate collected in step 3) of Example 1 is subjected to 0.1% SDS-12% polyacrylamide gel electrophoresis (120 volts, 1-2 Hour). Remove the gel and stain with 0.1M KCl until a transparent band appears on the gel block. Cut the transparent area where VhdQ is located with a scalpel, and place it in a dialysis bag (MWCO: 10000 Dalton, purchased from Shanghai Bioengineering Corporation), 120 volts for protein level electrophoresis. After 2 hours, the dialysis bag was taken out and put into pre-cooled 1000ml PBS for dialysis at 40C. Change to fresh PBS after every 5 hours for a total of three changes. After the dialysis is completed, the dialysis bag is taken out, the glue block inside is discarded, and the solution is carefully sucked out, which is the solution containing the recombinant VhdQ. The solution was centrifuged (6000 g, 4° C.) for ...

Embodiment 3

[0071] Example 3 Immunization application of recombinant VhdQ

[0072] Step 1) Preparation of recombinant subunit VhdQ vaccine antigen mixture and recombinant subunit VhdQ enhanced immune mixture. Culture strain B187 in LB medium at 300C to OD 600 0.8, and then centrifuged (5000g, 4°C) for 10min. Collect the cells and suspend them in PBS to a final concentration of 2×10 8 cfu / ml, namely B187-PBS. Mix 10 ul (concentration: 25 mg / ml) of the recombinant cross-protective vaccine antigen VhdQ obtained in Example 2 above with 1 ml of B187-PBS to obtain the recombinant subunit VhdQ vaccine antigen mixture. Mix 10 ul (concentration: 25 mg / ml) of the recombinant cross-protective vaccine antigen VhdQ obtained in Example 2 above with 1 ml of PBS to obtain the recombinant subunit VhdQ enhanced immune mixture. The strain B187 is preserved in CGMCC, General Microorganism Center of China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC No.2331.

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Abstract

The invention relates to the fields of molecular biology and genetic engineering, in particular to an intersecting protective vaccine antigen, a preparation method and an application thereof. The intersecting protective vaccine antigen is provided with a base sequence in a sequence table of SEQ ID NO. 1. The preparation method comprises the following steps of: establishing pET258 plasmid and then taking Vibrio Harveyi T4 as a moulding board; using primer VHQF5 and VHQR5 to carry out PCR augmentation and obtaining plasmid pBTQ connection established by products; transferring colon bacterium and obtaining plasmid pEVQ; and then carrying out inducement and obtaining antigen VhdQ. The intersecting protective vaccine antigen and B187-PBS culture fluid are mixed as the intersecting protective vaccine antigen of Vibrio Harveyi and vibrio parahaemolyticus. The vaccine antigen obtained by the preparation method exists in a plurality of pathogenicity vibrios, and has high conservative property, thus having intersecting protective effect. A highly active procaryon expression system obtained by the intersecting protective vaccine antigen can be used for expressing the intersecting protective vaccine antigen in large quantity; and the prepared antigen protein is guaranteed to have native immunity activity, and can be directly used for immune prophylaxis and treatment.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a cross-protective vaccine antigen and its preparation method and application. Background technique [0002] Vibrios are Gram-negative bacilli that are widely distributed throughout the world, especially in marine environments. The genus includes a variety of human and farmed aquatic organism pathogenic bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio harveyi, etc. Vibrio harveyi (Vibrioharveyi) has long been an important pathogen of shrimp, and its infection and spread have caused huge economic losses to the shrimp industry in my country. In addition to shrimp, Vibrio harveyi can also infect a variety of fish and shellfish; recent investigations and studies have shown that this bacteria has become one of the main pathogens that endanger the development of mariculture in my country, especially in the north, in recent ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/28C12N15/31C12N15/70A61K39/106A61K35/74A61P31/04
Inventor 孙黎张卫卫程爽
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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