CN100396778C

CN100396778C - Construction of secretory coenosarcus plasmid for bombyx mori rhabditis viral expression system

Info

Publication number: CN100396778C
Application number: CNB2006100493930A
Authority: CN
Inventors: 吴小锋; 姚慧鹏; 王冰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2006-01-26
Filing date: 2006-01-26
Publication date: 2008-06-25
Anticipated expiration: 2026-01-26
Also published as: CN1818072A

Abstract

The present invention discloses a construction method for a secretory donor plasmid of a bombyx mori baculovirus expression system. The construction of the plasmid is based on the existing donor plasmid pFastBacHTa, b, c and a baculovirus transfer carrier pAcGP67-B at present. The present invention is constructed by a gene recombination principle, and has the characteristic of strong protein secretion ability. The construction of the secretory donor plasmid can create conditions for recombinant protein of obtained secretion.

Description

The construction process of the secretor type donor plasmid of baculovirus expression vector system

Technical field

The present invention relates to genetic engineering technique, baculovirus gene expression system, protein purification and activation analysis, relate in particular to a kind of construction process of secretor type donor plasmid of baculovirus expression vector system.

Background technology

The silkworm baculovirus gene expression system is one of eukaryotic expression system efficiently at present, but present technology recombinant virus make up and purge process in exist the shortcoming that recombination fraction is low, the plaque analytical technology is loaded down with trivial details, spended time is grown.In order to address this problem preferably, we use for reference external AcNPV rapid gene expression system principle of work, utilize the assignment of genes gene mapping transferance of bacterial transposon, in intestinal bacteria, realize the transfer reorganization of gene, obtain recombinant virus fast, (patent is authorized to have made up the rapid gene expression system that can utilize China characteristic resources insect one silkworm, title: utilize bacterial transposon to make up the method Granted publication day of silkworm virus rapid gene expression system: 2005-10-11, the patent No.: 2003101087818).This system by donor plasmid, contain the genomic competence bacterium of silkworm baculovirus Bacmid DH10BmBac and constitute, its principle that produces recombinant virus is: at first the external source goal gene is cloned into donor plasmid, donor plasmid is transformed in the DH10BmBac competent cell then, fixed point transposition effect by bacterial transposon, promotor and goal gene are changed over to the silkworm baculovirus genome in the lump and produce recombinant virus dna, last transfection silkworm cultured cell and obtain to contain the goal gene recombinant virus.Donor plasmid is that the decision goal gene is the important tool how to express, American I nvitrogen company existing procucts are sold at present, name is called pFastBacHTa, b, c (catalog number: 10584-027), but this plasmid does not contain protein signal peptide secretion signal, causes the recombinant expressed of expression can't secrete the extracellular.And baculovirus transferring plasmid pAcGP67-B (BD Biosciences company: catalog number (Cat.No.): 21223P) contain a strong signal peptide sequence that can instruct protein secreting, name is called GP67 Signal Sequence.Utilize the gene recombination principle,, this signal peptide sequence is recombined into pFastBacHTa, b, c donor plasmid by gene manipulation techniques.The secretor type donor plasmid of Huo Deing can be used in the quick baculovirus expression system of silkworm like this.

Summary of the invention

The object of the present invention is to provide the construction process of the secretor type donor plasmid of the quick baculovirus expression system of a kind of silkworm.The structure of this plasmid is based on present existing donor plasmid pFastBacHTa, b, and c and baculovirus transfer vector pAcGP67-B adopt the gene recombination principle to make up and form, and having strong protein secreting ability is feature.Being configured to of this secretor type donor plasmid can obtain the excretory recombinant protein and create condition.

In order to achieve the above object, its step of the technical solution used in the present invention is as follows:

Baculovirus transferring plasmid pAcGP67-B has strong secreting signal peptide gp67 sequence in polyhedron promotor Ph downstream, this sequence is given recombinant protein and is had the outer ability of very strong secretion born of the same parents, the pAcGP67-B plasmid is transformed people intestinal bacteria DH5 competent cell respectively, carry out plasmid extraction subsequently, and carry out EcoRV and HindIII double digestion and handle, separate the gene fragment that obtains to have polyhedron promotor Ph promotor, gp67 signal peptide sequence and multiple clone site, size is 377bp; And to existing donor plasmid pFastBacHTa, b, c carries out SnaBI and the HindIII enzyme is cut processing, excise original polyhedron promotor Ph promotor and multiple clone site thereof, because EcoRV and SnaBI are flat terminal, therefore the fragment of above-mentioned 377bp can be linked to each other with above-mentioned fragment, produce a new donor plasmid.

The useful effect that the present invention has is: the structure of this plasmid is based on present existing donor plasmid pFastBacHTa, b, c and baculovirus transfer vector pAcGP67-B adopt the gene recombination principle to make up and form, and having strong protein secreting ability is feature.Being configured to of this secretor type donor plasmid can obtain the excretory recombinant protein and create condition.

Description of drawings

Fig. 1 is baculovirus transfer vector pAcGP-B figure;

Fig. 2 is donor plasmid pFastBacHTa, b, c figure;

Fig. 3 is that the secretor type donor plasmid makes up synoptic diagram;

A. baculovirus transfer vector pAcGP67-B restriction enzyme site synoptic diagram;

B. donor plasmid pFastBacHTa, b, c (Invitrogen), MCS among the figure (Multiple Cloning Sites) represents multiple clone site.

Embodiment

In order to make recombinant protein secrete effectively outside the born of the same parents, made up donor plasmid with strong secretion capacity characteristic.Baculovirus transferring plasmid pAcGP67-B (BDBiosciences company: catalog number (Cat.No.): 21223P, Fig. 1) have strong secreting signal peptide gp67 sequence in polyhedron promotor Ph downstream, this sequence is given recombinant protein and is had the outer ability of very strong secretion born of the same parents, the secretion of pAcGP67-B plasmid is transformed people intestinal bacteria DH5 competent cell, carry out plasmid extraction subsequently, and carry out EcoRV and HindIII double digestion and handle, separate the gene fragment that obtains to have Ph promotor, gp67 signal peptide sequence and multiple clone site, size is 377bp; And to existing donor plasmid pFastBacHTa, b, c (American I nvitrogen company product, catalog number (Cat.No.): 10584-027, Fig. 2) carry out SnaBI and the HindIII enzyme is cut processing, excise original Ph promotor and multiple clone site thereof,, therefore the fragment of above-mentioned 377bp can be linked to each other with above-mentioned fragment because EcoRV and SnaBI are flat end, produce a new donor plasmid, with its called after pBmBacGP67a, b, c.Operating process sees Fig. 3 for details.

1, research material: (authorize by patent by this research department's structure for the quick baculovirus expression system of silkworm, title: utilize bacterial transposon to make up the method Granted publication day of silkworm virus rapid gene expression system: 2005-10-11, the patent No.: 2003101087818) make up voluntarily by the applicant.DH5 α, DH10 β bacterial strain, donor plasmid pFastBacHTa, b, c are all available from American I nvitrogen.Baculovirus transfer vector pAcGP67-B is available from BD company.Various restriction enzymes, ligase enzyme equimolecular biologic operation reagent are available from Japanese Takara company.

2, workflow:

The secretion of pAcGP67-B plasmid is transformed people intestinal bacteria DH5 competent cell, carry out plasmid extraction subsequently, and carry out EcoRV and the processing of HindIII double digestion, and separate the gene fragment that obtains to have Ph promotor, gp67 signal peptide sequence and multiple clone site, size is 377bp; And to existing donor plasmid pFastBacHTa, b, c (American I nvitrogen company product, catalog number (Cat.No.): 10584-027, Fig. 2) carry out SnaBI and the HindIII enzyme is cut processing, excise original Ph promotor and multiple clone site thereof,, therefore the fragment of above-mentioned 377bp can be linked to each other with above-mentioned fragment because EcoRV and SnaBI are flat end, produce a new donor plasmid, with its called after pBmBacGP67a, b, c.Operating process sees Fig. 3 for details.

Claims

1. the secretor type donor plasmid pBmBacGP67a of baculovirus expression vector system, b, the construction process of c, it is characterized in that: the secretion of pAcGP67-B plasmid is transformed people intestinal bacteria DH5 competent cell, carry out plasmid extraction subsequently, and carry out EcoRV and the processing of HindIII double digestion, and separate the gene fragment that obtains to have polyhedron promotor Ph promotor, gp67 signal peptide sequence and multiple clone site, size is 377bp; And to existing donor plasmid pFastBacHTa, b, c carry out SnaBI and the HindIII enzyme is cut processing, excise original polyhedron promotor Ph promotor and multiple clone site thereof, and link to each other, produce a donor plasmid pBmBacGP67a with the fragment of above-mentioned 377bp, b, c.