Insect bioreactor expressing multiple exogenous genes and its construction method and application

A bioreactor and construction method technology, applied in the field of bioreactor construction, can solve the problems of low expression efficiency, complex, cumbersome process, etc., and achieve the effect of increasing expression amount and optimizing expression

Active Publication Date: 2011-12-21
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

However, this method introduces a single-copy DNA replication origin into the AcNPV genome, and only one copy of the AcNPV genome can be replicated in one Escherichia coli cell, resulting in low yield and poor quality of baculovirus genomic DNA, and low transfection efficiency of subsequent insect cells
At the same time, it uses the principle of transposition to transfer foreign genes and resistance selection markers to the LacZ gene, and the process of screening without forming blue spots is cumbersome, complicated, and the expression efficiency is low.

Method used

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  • Insect bioreactor expressing multiple exogenous genes and its construction method and application
  • Insect bioreactor expressing multiple exogenous genes and its construction method and application
  • Insect bioreactor expressing multiple exogenous genes and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0057] Experimental example 1 Construction of bacmid containing high copy replication initiator

[0058] 1 Experimental method

[0059] A targeting vector containing a high-copy replicon gene and homology arms of cat gene and chi gene at both ends was constructed.

[0060] 1.1. About the configuration of solution and medium (Joseph et al., Molecular Cloning Experiment Guide, Third Edition, 2002; Osper et al., Refined Molecular Biology Guide, 1998)

[0061] Solution I: 50mmol / L glucose, 25mmol / L Tris-HCl (pH8.0), 10mmol / L EDTA.

[0062] Solution II: 0.2mol / L NaOH, 1% SDS (prepared and used immediately).

[0063] Solution III: 100mL system, 5mol / L potassium acetate 80mL, glacial acetic acid 12mL, ddH 2 O8mL. TAE (50×): 242g Tris base, 57.1mL glacial acetic acid, 100mL 0.5mol / L EDTA (pH8.0), dilute to 1000mL with sterile water.

[0064] TER solution: Pancreatic RNAse (RNAse A) was dissolved in 10mM Tris-HCl, 15mMNaCl, made into a 10mg / mL stock solution and stored at -20°C, t...

experiment example 2

[0174] Experimental Example 2: Introducing high-copy replicons into the genomes of silkworm BmNPV and AcNPV to obtain BmBacmid and AcBacmid

[0175] 2.1 Preparation of Escherichia coli BW25113 Competent for Electrical Stimulation Containing Red Recombinase

[0176] The red recombinase gene exists on the PKD46 plasmid in the BW25113 strain. There is a repA101(ts) gene on this plasmid, which will lose the plasmid when cultured at 37°C. The recombinase gene is expressed under the induction of L-arabinose, therefore, the preparation process of this electric shock competence is as follows:

[0177] (1) Pick a monoclonal colony with a sterilized toothpick, inoculate it into 4ml LB liquid medium at 37°C, 250rpm, and culture overnight with shaking.

[0178] (2) The above bacterial solution was inoculated into 400ml LB liquid medium at a ratio of 1:100, cultured with shaking at 37°C and 250rpm for about 1.5-2h until the OD600 value of the bacterial solution reached about 0.3, and L-ar...

experiment example 4

[0220] Experimental Example 4 The reverse selection marker gene is introduced into the P10 gene locus in BmBacmid and AcBacmid

[0221] The promoter of the P10 gene is a high-efficiency promoter, which is suitable for expressing foreign proteins. Therefore, the BmBacmid and AcBacmid with the deletion of the P10 gene and the reverse marker gene were constructed here by the method of reverse selection.

[0222] 4.1 Synthesis of P10 gene knockout vector

[0223] Synthesize the pP10 vector backbone with P10 gene homology arms and the pP10 vector backbone containing multiple cloning restriction sites in the middle, and the P10 vector backbone consists of three parts ( Figure 6 shown); Figure 6 In the middle, the black parts on both sides are the enzyme cutting sites, which are used for cutting out the gene on the T vector. The upstream and downstream are EcoRV, BstXI, HindIII and Sal I, Spe I, Cla I enzyme cutting sites, and the white part is P10 The homology arm of the gene, t...

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Abstract

The invention discloses an insect bioreactor capable of expressing multiple exogenous genes, and a construction method and application thereof. The construction method comprises the following steps: (1) introducing multicopy high-efficiency bacteria DNA (deoxyribonucleic acid) replication initiator into chitinase and cysteine proteinase genes of a baculovirus genome to obtain a baculovirus shuttle plasmid; (2) replacing virus duplicated essential gene downstream the polyhedrosis gene of the baculovirus shuttle plasmid with antibiotic gene to obtain a baculovirus plasmid DNA; and (3) replacingother virus duplicated and infected nonessential genes in the baculovirus shuttle plasmid with reverse selection marker gene to obtain the insect bioreactor. The antibiotic gene or reverse selection marker gene in the insect bioreactor which is constructed by replacing the exogenous target genes can express multiple exogenous genes in a host insect or insect cell. The insect bioreactor disclosed by the invention can efficiently expressing one or more exogenous genes in an insect body at the same time, and can produce massive recombinant proteins at low cost.

Description

technical field [0001] The present invention relates to a method for constructing a bioreactor for expressing exogenous target genes, in particular to a method for constructing an insect baculovirus bioreactor capable of efficiently expressing a variety of exogenous target genes in insects, and the method constructed by the method The product belongs to the field of construction of bioreactors expressing exogenous genes. Background technique [0002] The baculovirus expression system, especially the silkworm (Bm)-bombyx mori baculovirus (BmNPV) expression system, is one of the most commercially exploitable eukaryotic individual expression systems in the world. It has become one of the four major expression systems commonly used in genetic engineering today (ie, baculovirus, Escherichia coli, yeast, and mammalian cell expression systems). [0003] The bioreactor of the baculovirus expression system was established in the 1980s. Since the first high-efficiency expression of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/63C12N7/01
Inventor 张志芳李轶女易咏竹韦永龙刘兴健沈桂芳舒惠国
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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