Method of preparing ocean double RNA virus MABV recombination albumen and application thereof

An RNA virus, marine technology, applied in the field of genetic engineering to achieve the effect of increasing credibility

Inactive Publication Date: 2007-08-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there are no patents and reports on the use of genetic engineering methods to prepare virus coat proteins as standard proteins, as immune vaccines, and as antigens to prepare antibodies

Method used

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  • Method of preparing ocean double RNA virus MABV recombination albumen and application thereof
  • Method of preparing ocean double RNA virus MABV recombination albumen and application thereof
  • Method of preparing ocean double RNA virus MABV recombination albumen and application thereof

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Embodiment Construction

[0039] Isolation of MABV virus and cloning of VP2 and VP3 genes

[0040] The MABV isolated from the susceptible sweetfish (Plecoglossus altivelis) was cultured in the salmon cell line CHSE-214 (Chinook salmon embryo cell line) at 20°C, the virus was propagated, and the viral RNA was further extracted and reverse-transcribed into cDNA .

[0041] Primers were designed using cDNA reverse-transcribed from total RNA of fish cells infected with MABV Y-6 virus as a template. The forward primer contains a BamHI restriction site (underlined bases), and the reverse primer contains an XhoI restriction site (underlined bases).

[0042] vp2 forward primer (5'-gg atcc atgtccctcaccacg-3')

[0043] vp2 reverse primer (5'- ctcgag ttaggccaccagggtg-3)

[0044] vp3 forward primer (5'- ggatcc tcagggatggatgaagag-3')

[0045] vp3 reverse primer (5'- ctcgag ttacacttctccgttatctcc-3')

[0046] PCR amplification conditions: 94°C for 1min, 54°C for 40s, 72°C for 1min, 30 cycles. After PCR am...

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Abstract

The invention discloses a preparing method of restructuring protein with ocean double RNA virus MABV structural protein gene, which is characterized by the following: the restructuring protein includes VP2 fusing protein or VP3 fusing protein and VP2 or VP3 protein; the VP2 fusing protein coded sequence possesses SEQ ID NO.1 nucleic acid sequence in sequence table; the VP3 fusing protein coded sequence possesses SEQ ID NO.2 nucleic acid sequence in sequence table; the polyclonal antibody produced by reconstructing protein increases confidence level during testing ocean double RNA virus MABV; it uses ELISA and western blot to test and is simpler than other method (RT-PCR, original position and so on). To set VP2 and VP3 as vaccine is safer than attenuated vaccine.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion protein containing a marine double RNA virus coat protein gene VP2 or VP3 gene, a preparation method and an application. Background technique [0002] The genus Aquabirnavirus (Aquabirnavirus) belongs to the family Birnaviridae (Birnaviridae), and an obvious feature of this family is that the viral genome is composed of double-segment double-stranded RNA. The representative species of the Aquabirnavirus genus is Infectious Pancreatic Necrosis Virus (IPNV), which can cause acute infectious disease in salmon. Sorimachi M, Hara T. In 1985, a marine double RNA virus was first isolated from a yellowtail (Seriolaquinqueradiana) (Sorimachi M, Hara T. "Characteristics and pathogenicity of a virus isolated from yellowtailfingerlings showing ascites[J]" "Fish Pathol" 1985 , 19:231-238.), the typical symptoms of the disease are anemia, gill necrosis, hepatic hemorrhage, ascites,...

Claims

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Application Information

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IPC IPC(8): C12N15/33C12N1/21C07K19/00C07K1/14C07K14/08A61K39/12C07K16/18G01N33/53G01N33/569
Inventor 张传溪徐海君杨章女林建国
Owner ZHEJIANG UNIV
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