II-set alphabaculovirus Bacmid capable of expressing foreign protein in various insect cells and application thereof

A technology for exogenous protein and baculovirus, applied in the fields of molecular biology and bioengineering, can solve the problems of time-consuming, complicated operation of plaque purification, and low yield of recombinant virus, and achieve the effect of maintaining complete characteristics

Active Publication Date: 2015-10-28
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
View PDF5 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low production rate of recombinant viruses and the complicated and time-consuming operation of plaque purification, it often takes several months or even longer to construct a complete recombinant virus

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • II-set alphabaculovirus Bacmid capable of expressing foreign protein in various insect cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The construction of a group A baculovirus Mb Bacmid expressing foreign proteins in various insect cells comprises the following steps:

[0036] (1), construction of the cabbage armyworm nuclear polyhedrosis virus Bacmid transfer vector pMbTV

[0037] A bacterial DNA replicon (mini-F replican) containing a low copy number, a kanamycin resistance gene and a lacZα gene, a short fragment inserted into the lacZα gene as a bacterial transposon insertion site (mini-attTn7) N-terminal, forming a DNA fragment of 8.6 kb. This fragment is preceded by a fragment of about 2.3kb upstream of the polyhedrin gene of Brassica cabbage nuclear polyhedrosis virus as an upstream homology arm, followed by an approximately 1.3kb downstream sequence of the polyhedrin gene as a downstream homology arm, forming a transfer vector pMbTV ( figure 1 ). The transfer vector construction process is:

[0038] First, two pairs of primers were designed according to the genome sequence of Brassica olera...

Embodiment 2

[0046] An application of group A baculovirus Mb Bacmid expressing foreign proteins in various insect cells, comprising the following steps:

[0047] (1) Construction of a donor plasmid containing the reversion gene PH and the early expression pie1+cath gene of the virus

[0048] The primers of viral polyhedrin gene (comprising promoter sequence), viral early gene 1 promoter sequence (pie1) and viral cathepsin gene cath are designed respectively according to the whole genome sequence of cabbage armyworm nuclear polyhedrosis virus, and primer sequence is: Ph-1:5'-CC GGATCC CTATTAGTAGTAAACTGTATCGTG-3' (the underline is the BamH I restriction site), Ph-2:5'-GC GTATAC TTAATATGCAGGACCAGTGTATAG-3' (the underline is the BstI endonuclease site); Pie1-1:5'-A GATGTC CATACCTGTCAGGGAATGC-3' (the underline is the Bgl endonuclease site), Pie1-2:5'-GG ATCCCG AGTTATTAGCGTCATAGCCTTCC-3' (the underline is the BamH I endonuclease site); Pcath-1: 5'-G GAATTC TGTATGCTCGTTACAGTTACAGC-3' (th...

Embodiment 3

[0059] An application of group A baculovirus Mb Bacmid expressing foreign proteins in various insect cells, comprising the following steps:

[0060] (1), Containing the construction of reversion gene Ph and p10 promoter donor plasmid pFastBac

[0061] Using the PCR method, the protein gene of the cabbage spodoptera nuclear polyhedrosis virus with promoter sequence was amplified from the virus genome, and cloned into pGEM T-Easy to form the pGEM T-Easy PH plasmid. At the same time, the viral p10 gene promoter was amplified and cloned into pGEM T-Easy to form pGEM T-Easy p10 plasmid. Construction According to the appropriate endonuclease site, connect the polyhedrin gene with the promoter and the p10 promoter to form a fragment containing the double promoters of the PH gene and the p10 gene, and construct a donor containing the reversion gene Ph and the p10 promoter Plasmid pFastBac. The specific method is:

[0062] First, select a sequence located 190 bp upstream of the star...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a II-set alphabaculovirus shuttle expression vector MbBacmid capable of expressing foreign protein in various insect cells and application thereof. The Bacmid contains whole genome sequences of mamestra brassicae nuclear polyhedrosis viruses, and contains genetic elements capable of being copied in escherichia coli. Foreign protein genes can be quickly inserted into the Bacmid provided by the invention through a donor plasmid pFast-Bac Mbph, and are efficiently expressed inside various commercial insect cells including Sf9, Tn368, high five and the like. The vector and a donor plasmid pFast-Bac Mbph+piel-cath can be subjected to homologous recombination to obtain a recombinant virus MbMNPV-ph+ piel-cath capable of increasing insect killing speed. The expression system provided by the invention is the only II-set alphabaculovirus shuttle expression vector system capable of efficiently expressing foreign protein in various commercial insect cells.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and bioengineering, and specifically relates to a group II type A baculovirus Bacmid expressing foreign proteins in various insect cells and its application. Background technique [0002] Baculovirus is a common name for members of the Baculoviridae family. According to the 9th report published by the International Committee on Taxonomy of Viruses (ICTV) in 2012, the Baculoviridae is divided into four genera, which are the genus A and Baculoviridae. [Alphabaculovirus, a nucleopolyhedrovirus (NPV) that infects Lepidoptera insects], Beta baculovirus [Betabaculovirus, a Granulovirus that infects Lepidoptera insects (Granulovirus, GV)], Type C Gammabaculovirus (NPV that infects Hymenoptera) and Deltabaculovirus (NPV that infects Diptera). According to the sequence characteristics of viral membrane fusion protein and DNA polymerase protein and other characteristics of the virus, type A bacu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/866C12N7/01C12R1/19
Inventor 张忠信吴柳柳类承凤周吟程丹凝刘柳
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap