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Recombinant baculovirus expression vector of ORF66 protein of cyprinid herpesvirus II and preparation method thereof

A technology of recombinant baculovirus and ORF66 is applied in the field of recombinant baculovirus expression vector of carp herpes virus type II ORF66 protein and its preparation, which can solve the problems of false positive, template contamination, high detection cost, etc., and achieve high-efficiency expression and storage powerful effect

Pending Publication Date: 2019-11-05
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although molecular biology detection technology has high sensitivity, it is also easy to cause false positives due to template contamination, so it cannot be used as the only standard for diagnosing CyHV-2 infection
On the other hand, techniques such as molecular biology require expensive instruments and equipment, high testing costs, and high technical requirements for testing personnel, making them unsuitable for rapid on-site testing and popular application at the grassroots level.

Method used

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  • Recombinant baculovirus expression vector of ORF66 protein of cyprinid herpesvirus II and preparation method thereof
  • Recombinant baculovirus expression vector of ORF66 protein of cyprinid herpesvirus II and preparation method thereof
  • Recombinant baculovirus expression vector of ORF66 protein of cyprinid herpesvirus II and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Construction of recombinant baculovirus vector containing CyHV ORF66 gene and acquisition of recombinant shuttle bacmid rBacmid-CyHV-ORF66.

[0036] 1.1 It independently designed primers with reference to the full-length sequence of the ORF66 gene in the CyHV-2SY-C1 strain (Access No. KM200722.1) published on GenBank, and used the extracted CyHV-2 cDNA as a template to amplify the ORF66 gene, in which the upstream primer ORF66 was added The sequence after the BamH1 restriction endonuclease site is:

[0037] SEQ ID NO: 3, after the downstream primer is added to the HindIII restriction site, the sequence is:

[0038] SEQ ID NO: 4, the underlined part is the restriction site, using the extracted CyHV-2 cDNA as a template, the full-length gene of ORF66 was amplified, about 1200bp, which was consistent with the expected size. PCR results such as figure 1 shown.

[0039] 1.2 Construction of recombinant baculovirus vector pFastBac HTA-CyHV-ORF66.

[0040] T...

Embodiment 2

[0042] Example 2: Expression of Cyprin herpesvirus type II ORF66 protein in insect cells.

[0043] 2.1 Transfection of insect cells

[0044] (1) Take 2 mL of well-grown Sf9 insect cells and count about 2×10 6 Add cells into a six-well plate, and allow the cells to adhere to the wall for 3 hours;

[0045] (2) Prepare two 1.5mL sterilized centrifuge tubes and mark them well. Add 5 μL Bacmid-CyHV-ORF66 and 100 μL Grace medium without antibiotics and FBS to one centrifuge tube and mix gently; another centrifuge tube Mix 6 μL of Cellfectin Reagent and 100 μL of Grace medium without antibiotics and FBS in the medium; Cellfectin Reagent is fat-soluble, and precipitation will appear when left for a long time, so it is best to shake it evenly before use;

[0046] (3) The solutions in the two tubes were mixed and incubated at room temperature for 45 minutes;

[0047] (4) Add 800 μL of Grace medium. Then the Grace medium in the six-well plate was removed, washed once gently with Grac...

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Abstract

The invention provides a recombinant baculovirus expression vector of an ORF66 protein of cyprinid herpesvirus II and a preparation method thereof. An ORF66 gene is amplified with extracted CyHV-2 cDNA as a template, the amplified ORF66 gene is connected to a baculovirus vector pFastBacHTA, a recombinant baculovirus vector pFastBac HTA-CyHV-ORF66 is constructed and is transformed into an escherichia coli competent cell DH10Bac, and a recombinant shuttle bacmid rBacmid-CyHV-ORF66 is obtained; after being identified to be correct, the recombinant shuttle bacmid rBacmid-CyHV-ORF66 is transfectedto an Sf9 insect cell, and a recombinant baculovirus is obtained; the recombinant baculovirus is subjected to passage amplification, the baculovirus containing the CyHV-2-ORF66 gene and having high titer is inoculated into the sf9 insect cells, and eukaryotic expression of a CyHV-2-ORF66 protein is carried out. The method is selected for constructing the recombinant baculovirus expression vector of the ORF66 protein of the cyprinid herpesvirus II, and the recombinant baculovirus expression system is used for expressing the ORF66 protein of the cyprinid herpesvirus II in the insect cell, provides materials for preparation of a specific monoclonal antibody of the ORF66 protein of the cyprinid herpesvirus II, and lays a foundation for a cyprinid herpesvirus serological ELISA detection method.

Description

technical field [0001] The invention relates to the field of gene recombination expression in biotechnology, in particular to a recombinant baculovirus expression vector of carp herpesvirus type II ORF66 protein and a preparation method thereof. Background technique [0002] Cyprinid herpesvirus type Ⅱ (Cyprinid herpesvirus 2, CyHV-2) is the pathogen that causes hematopoietic organ necrosis in crucian carp. The virus belongs to Herpesvirales, Alloherpesviridae, Cyprinivirus , CyHV-2 is closely related to two other viruses, Cyprinid herpesvirus 1 (CyHV-1) and Cyprinid herpesvirus 3 (CyHV-3), which are isolated from carps. Ictalurid herpes-virus 1 (IcHV-1) is relatively distantly related. In terms of etiology, CyHV-2 has the general characteristics of fish herpesviruses. The virus is spherical and has a capsid structure with icosahedral stereosymmetry, which contains a double-stranded DNA with a length of about 290,304 bp inside, and a cortical layer and a capsule on the out...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N15/66C12N15/38
CPCC12N15/86C12N15/66C07K14/005C12N2710/14043C12N2710/16022
Inventor 李强栾林林
Owner YANCHENG INST OF TECH
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