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Pseudotype insect baculovirus gene transfer system for prawns, virus, construction method and application

A baculovirus and gene transfer technology, applied in the field of genetic engineering, can solve the problems of low philotropy of prawn cells, hindering the smooth development of transgenic prawns and prawn gene editing research, and inability to effectively infect prawn cells, etc.

Active Publication Date: 2020-07-07
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, previous studies have shown that the insect baculovirus expression system cannot effectively infect shrimp cells, indicating that its tropism in shrimp cells is extremely low, and it cannot be applied to gene transfer studies in shrimp tissues and cells
[0005] At present, there is still a lack of an efficient gene transfer and expression technology in shrimp adults and in vitro cultured cells, which seriously hinders the smooth development of transgenic shrimp and shrimp gene editing research

Method used

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  • Pseudotype insect baculovirus gene transfer system for prawns, virus, construction method and application
  • Pseudotype insect baculovirus gene transfer system for prawns, virus, construction method and application
  • Pseudotype insect baculovirus gene transfer system for prawns, virus, construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Cloning and modification of the coding frame nucleic acid sequence of the envelope protein gene (VP28) of shrimp WSSV virus and the construction of its eukaryotic expression vector pcDNA3.1-VP28.

[0057] Genomic DNA of WSSV-infected prawns was extracted using the DNA extraction kit (Easypure Marine Animal Genomic DNAkit, Cat. No. EE151-01) of Quan Shi Jin Company, and the specific operation method was carried out according to the kit instructions. The transformation of the nucleic acid sequence of the coding frame of the shrimp virus VP28 gene was introduced by means of amplification primers, that is, VP28 gene-specific primers containing BamHI and EcoRI restriction sites were designed respectively: the sequence of the forward mutation primer was 5’-CGC GGATCC ATTGCCACCATGGATCTTTCTTTCAC-3' (SEQ ID NO.5), reverse mutation primer sequence is 5'-CCG GAATTC GTTACTCGGTCTCAGTGCC-3' (SEQ ID NO. 6). The target fragment was amplified by PCR technique. The system and procedu...

Embodiment 2

[0060] The foreign gene was cloned into the donor plasmid pFastBac1.

[0061] Take the construction of pFastBac1-GUS recombinant plasmid as an example ( figure 2 , the preparation diagram of the recombinant bacmid-GUS plasmid; among them, i is the electrophoresis result of pFastBac1-GUS plasmid DNA, ii is the blue and white spot screening result of the recombinant bacmid-GUS, and iii is the recombinant bacmid Bacmid -GUS extraction and electrophoresis results).

[0062] GUS gene-specific primers containing BamHI and EcoRI restriction sites were designed respectively: the forward primer sequence is 5'-CGC GGATCC ATGGTCCGTCCTGTAGAAAC-3' (SEQ ID NO.7), the reverse primer sequence is 5'-CCG GAATTC TCATTGTTTGCCTCCCTGCT-3' (SEQ ID NO. 8). The target fragment was amplified by PCR technique. The system and procedure of the PCR reaction are as follows: 50 μL of the system consists of: 25 μL 2×Hieff PCRMasterMix (containing Mg 2+ ), 2.5 μL 10 μmol / L upstream and downstream prime...

Embodiment 3

[0065] The exogenous gene is recombined into the viral plasmid Bacmid.

[0066] Take the construction of Bacmid-GUS as an example ( figure 2 ).

[0067] The pFastBac1-GUS plasmid was transformed into DH10Bac competent cells. The transformation product was plated, and after blue-white screening and bacterial liquid PCR detection, the recombinant Bacmid plasmid containing the GUS gene was purified: Bacmid-GUS.

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Abstract

The invention relates to a pseudotype insect baculovirus gene transfer system for prawns, a virus, a construction method and an application, and belongs to the technical field of genetic engineering.The pseudotype insect baculovirus gene transfer system for prawns disclosed by the invention comprises a Bac-to-Bac insect baculovirus expression system, prawn virus envelope protein gene expression plasmids and insect packaging cells. The pseudotype insect baculovirus gene transfer system for prawns disclosed by the invention can stably and efficiently transfer and express exogenous genes in prawn tissue and cells.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a pseudotyped insect baculovirus gene transfer system for prawns, a virus and its construction method and application, that is, a pseudotyped insect baculovirus gene transfer system for prawns, a pseudotyped insect baculovirus gene transfer system for prawns, Pseudotyped insect baculovirus of prawn and its construction method and application in prawn adult tissues and prawn cells (in vitro cultured cells). Background technique [0002] The Bac-to-Bac insect baculovirus expression system (Invitrogen product) is a very mature insect cell gene transfer technology. The system consists of transfer plasmid, helper plasmid, baculovirus Bacmid plasmid and competent cell DH10Bac. It is based on the transposition principle of E. coli transposase Tn7, and successfully realizes the rapid recombination of foreign genes into baculovirus in E. coli Bacmid plasmid, and the purpose o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66A01K67/033
CPCC12N15/86C12N15/66A01K67/0338C12N2710/14043A01K2227/70A01K2267/02C12N2710/14052C12N2770/32022C12N2770/32045C07K14/005C12N7/00C12N2800/105
Inventor 郭华荣毋梦茜
Owner OCEAN UNIV OF CHINA
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