Construction method for linearized shuttle vector BmBacmid

A technology of shuttle vector and construction method, which can be used in biochemical equipment and methods, botanical equipment and methods, viruses/phages, etc., and can solve the problems of high-throughput expression of foreign genes.

Inactive Publication Date: 2012-07-11
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the silkworm baculovirus expression system based on nuclear polyhedrosis virus BmNPV has also developed linear...

Method used

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  • Construction method for linearized shuttle vector BmBacmid
  • Construction method for linearized shuttle vector BmBacmid
  • Construction method for linearized shuttle vector BmBacmid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the construction of pBacAvrII2 vector

[0047] The pBacAvrII2 vector was modified on the basis of the pBacPAK8 vector, and two AvrII sites were successfully introduced. Proceed in two steps as follows:

[0048] (1) The introduction of the first AvrII site to construct the pBacAvrII1 vector:

[0049]Perform site-directed mutation on the orf1629 gene flanking the 3′ end of the polyhedron gene promoter of the vector pBacPAK8DNA, and mutate TTTAGG into CCTAGG—that is, the AvrII site, so that it is introduced between SnaBI and SwaI, that is, at the 3′ end of the orf1629 gene The first AvrII restriction site. Primers were designed as follows:

[0050] P1: 5′-CGGTACGTATTTTAATAATTCATTAAATTTATAATC CCTAGG -3' (the underlined part is the AvrII site)

[0051] P2: 5'-GAGGATTTAATATTTAAATTCAG-3';

[0052] PCR amplification was performed using pBacPAK8 DNA as a template. The PCR reaction system is:

[0053]

[0054] The reaction program was: pre-denaturation a...

Embodiment 2

[0059] Embodiment 2, the construction of the pBACAvrII2 vector containing bacterial artificial chromosome (BAC)

[0060] Forward primer P3 and reverse primer P4 were synthesized according to the sequence of the BAC fragment (including mini-F, LacZa-attTn7 and Kanr) of the commercially available DH10Bac Bacmid (bMON14272) DNA (both P3 and P4 contain FseI restriction sites), the sequence as follows:

[0061] P3: 5′-TTGCTGATATC GGCCGGCC TTTTGCTTTGCCACGGAACGGTCTG-3′ (the underlined part is the FseI restriction site)

[0062] P4: 5′-CAGGATCGGCCTA GGCCGGCC TTGCCGGGTCCCAGGAAAGGAT-3' (the underlined part is the FseI restriction site)

[0063] Using the extracted shuttle carrier Bacmid DNA as a template, the PCR reaction system is:

[0064]

[0065]

[0066] Note: DH10Bac is a product of Invitrogen, and DH10Bac cells contain Bacmid vector and auxiliary plasmid pMON7124. KOD-FX refers to KOD-FX DNA polymerase.

[0067] The PCR reaction program was: pre-denaturation at 98°C...

Embodiment 3

[0068] Embodiment 3, the construction of the silkworm baculovirus shuttle vector BmBacmid

[0069] (1) Amplify the BAC fragment in the pBACAvrII2 vector

[0070] In order to enable the homologous recombination of the BAC fragment with the linearized BmBacPAK, the BAC fragment containing the flanking sequences of the polyhedron gene at both ends was amplified from pBACAvrII2, and the primers were designed as follows: P5: 5'-ACAAACTGGAAATGTCTATCAATATATAG-3' (including EcoRV site) and P6: 5′-ACACGTTAAATAAAGCTTGGACATATTTAACA-3′ (with HindIII site)

[0071] Using pBACAvrII2 as a template, use KOD-FX DNA polymerase to PCR amplify and synthesize BAC fragments. The PCR reaction system is:

[0072]

[0073] The reaction program is: pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds; annealing and extension at 68°C for 9 minutes, a total of 30 cycles, and finally extension at 68°C for 10 minutes. After the reaction, the PCR product was analyzed by 0.7% agaro...

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Abstract

The invention discloses a construction method for a linearized shuttle vector BmBacmid. The construction method comprises the following steps of: 1) constructing a pBacAvrII2 vector containing two AvrII loci; 2) constructing a pBACAvrII2 vector containing bacterial artificial chromosome (BAC), namely designing a pair of primers of which two ends contain FseI loci, amplifying a segment of the BAC on DH10BacBacmid, and inserting into the pBacAvrII2 vector to obtain the pBACAvrII2 vector; and 3) constructing the linearized shuttle vector BmBacmid, namely amplifying the segment of the BAC through polymerase chain reaction (PCR) by taking the pBACAvrII2 vector as a template, co-transfecting bombyxmori cells by using the PCR product and linearized BmBacPAK, performing homologous recombination in the bombyxmori cells to generate recombinant virus, thus obtaining the linearized shuttle vector BmBacmid.

Description

technical field [0001] The invention belongs to the technical field of carrier construction using genetic engineering methods in biotechnology. Background technique [0002] The silkworm is an important economic animal, which has the characteristics of easy breeding and low cost. The baculovirus expression system using silkworm as a host expresses foreign proteins with high yield, low cost and good quality, so it has broad market development prospects. Moreover, this expression system is safe, has a high expression level of exogenous genes, and can perform post-translational processing. It is an ideal eukaryotic expression system, so this system has been widely used. By transforming the baculovirus vector, the system has been continuously improved in the construction efficiency of the recombinant virus, the expression level and the expression flux of the foreign gene. Among them, the silkworm baculovirus expression system based on nuclear polyhedrosis virus BmNPV has also ...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/01
Inventor 陈健陈琴张耀洲吕正兵陈倩
Owner ZHEJIANG SCI-TECH UNIV
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