Preparation method of rabbit hemorrhagic fever virus empty capsid antigen

A rabbit hemorrhagic fever virus and virus technology, applied in the field of genetic engineering, can solve the problems of unoptimistic application prospect, unsatisfactory expression level, insolubility of VP60 and poor immune effect, and achieve low cost, low energy consumption and reduced production cost. Effect

Active Publication Date: 2012-01-04
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are still some problems in the research of RHDV genetically engineered vaccines: VP60 expressed by Escherichia coli has the disadvantages of insolubility and relatively poor immune effect, and

Method used

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  • Preparation method of rabbit hemorrhagic fever virus empty capsid antigen
  • Preparation method of rabbit hemorrhagic fever virus empty capsid antigen
  • Preparation method of rabbit hemorrhagic fever virus empty capsid antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1, Soluble expression of the original sequence VP60 of rabbit hemorrhagic fever virus capsid protein gene in silkworm bioreactor and detection of expression products

[0050] 1. Acquisition of the original sequence VP60 of the capsid protein gene of rabbit hemorrhagic fever virus

[0051] TRIzol method was used to extract genomic RNA from rabbit diseased tissues.

[0052] Primers were designed to amplify the original sequence VP60 (SEQ ID NO.1) of the capsid protein gene of rabbit hemorrhagic fever virus by RT-PCR. The designed reverse transcription specific primer is: RHDV-RT: 5'-GGATTAAAACCTAACCTACC-3'. The amplification primer of the original sequence VP60 of the capsid protein gene is: VP60 upstream: 5′-G GAATTC AACATGGAGGGCAAAGCCCGCACAG-3' (EcoR I); VP60 downstream: 5'-GA AGATCT TCAGACATAAGAAAGCCATTG-3' (Bgl II).

[0053] After PCR amplification, the fragment size was analyzed by agarose gel electrophoresis. The target gene fragment was recovered and...

Embodiment 2

[0078] Example 2, Soluble expression of capsid protein gene of rabbit hemorrhagic fever virus codon-optimized sequence VP60-O in silkworm bioreactor and detection of expression products

[0079] 1. Obtaining the optimized sequence VP60-O

[0080] According to the silkworm codon preference, the original sequence VP60 of the rabbit hemorrhagic fever virus capsid protein gene measured in Example 1 is optimized without changing the amino acid sequence. The optimized sequence VP60-O is shown in SEQ ID NO.2. In the original sequence Contains rare codons in tandem, which reduces the translation sequence or even untranslates the device. After optimization, the CAI value of the sequence is increased from 0.72 to 0.79. The GC content and unsuitable peaks are adjusted to prolong the half-life of mRNA. The GC content is adjusted from 54.9% to 53.34 %, those stem-loop structures that affect the stability of mRNA and its combination with ribosomes are destroyed, there is a Bam HI restrictio...

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Abstract

The invention relates to the field of genetic engineering and particularly relates to a preparation method of a rabbit hemorrhagic fever virus empty capsid antigen. The method provided by the invention comprises the following steps: 1) constructing a rhabdovirus transfer carrier containing a rabbit hemorrhagic fever virus capsid protein VP60 gene or an optimized gene, wherein codon optimization is performed according to the codon frequency of bombyx mori; 2) performing cotransfection on the constructed transfer expression carrier and DNA (deoxyribonucleic acid) of rhabdovirus so as to carry out homologous recombination or transposition to further obtain the recombinant rhabdovirus; 3) infecting the recombinant rhabdovirus with the host cells of an insect; and 4) culturing the infected host of the insect to express the corresponding rabbit hemorrhagic fever virus empty capsid antigen, and harvesting and purifying the expressed antigen. By adopting the method provided by the invention, the production cost of the rabbit hemorrhagic fever virus empty capsid antigen can be greatly reduced, and the method has a plurality of advantages of safety, high efficiency, low energy consumption, low cost and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method of rabbit hemorrhagic fever virus empty capsid antigen. Background technique [0002] Rabbit viral hemorrhagic disease, commonly known as rabbit plague, is an acute, virulent, highly contagious and fatal infectious disease caused by rabbit hemorrhagic disease virus (RHDV). It was once a devastating infectious disease of rabbits. , has attracted the attention of the rabbit industry because of bringing huge economic losses to the rabbit industry. The disease was first reported in China in 1984. In 1989, the World Organization for Animal Health (OIE) officially listed the disease as a Class B infectious disease, and my country classified it as a Class II infectious disease (Wang Yongkun et al., Diagnosis and Prevention of Rabbit Distemper, 1992). [0003] In the seventh report of the International Committee on Taxonomy of Viruses (ICTV) in 2000, rabbit hemor...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/866A01K67/033C07K14/08A61K39/12A61P31/14C12R1/93
Inventor 张志芳李轶女易咏竹王树坤石小峰王石宝张渭蛟舒惠国
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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