CN101381726A

CN101381726A - Method for expressing growth factor of human blood vessel in bombyx mori by Bac-to-Bac system

Info

Publication number: CN101381726A
Application number: CNA2008101216065A
Authority: CN
Inventors: 相兴伟; 吴小锋; 陈琳
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2008-10-14
Filing date: 2008-10-14
Publication date: 2009-03-11

Abstract

The invention discloses a method for expressing angiogenesis factors of human beings in domestic silkworms by a Bac-to-Bac system. The method comprises the following steps: recombinant viruses which contain the angiogenesis factors of endodermis of the human body are established by the Bac-to-Bac baculovirus expression system; and recombinant proteins are separated and purified from bodies of infected silkworms by a glycogen chromatography column. The method utilizes the Bac-to-Bac expression system, can quickly generate recombinant baculoviruses which contain the angiogenesis factors of the endodermis of the human body, highly efficiently expresses the recombinant baculoviruses in larva of the domestic silkworms, and utilizes the glycogen affinity chromatography column to realize quick separation and purification of the recombinant proteins.

Description

Method with Bac-to-Bac system expressing human angiogenesis factor in silkworm

Technical field

The present invention relates to biotechnology, baculovirus gene expression system, relate in particular to a kind of Bac-to-Bac of utilization baculovirus expression system is expressed human body interior cutaneous vessel somatomedin and purifying in silkworm method.

Background technology

The silkworm baculovirus gene expression system is one of eukaryotic expression system efficiently at present.The Bac-to-Bac baculovirus expression system that the applicant utilizes the bacterial transposon principle to make up to be specifically designed to silkworm is (referring to paper: Wu Xiaofeng, Cao Cuiping, Lu Xingmeng.2006, utilize bacterial transposon to make up the Bac-to-Bac rapid gene expression system that is applicable to silkworm.Silkworm industry science, 32 (2): 183-188).The advantage of this system is that recombinant baculovirus can be by the assignment of genes gene mapping transferance of bacterial transposon, in intestinal bacteria, realize the transfer reorganization of gene, obtain recombinant virus fast, made up the Bac-to-Bac rapid gene expression system that can utilize China characteristic resources insect-silkworm.This system by donor plasmid, contain the genomic competence bacterium of silkworm baculovirus BmBacmid 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.The recombinant virus that applicant's trial utilizes the quick generation of this system can express human body interior cutaneous vessel somatomedin, and utilize silkworm to express how to produce recombinant human interior cutaneous vessel growth factor protein, and utilize the affinity chromatography technology to set up and separate this proteic method as the host.

Summary of the invention

The invention discloses a kind of method with Bac-to-Bac system expressing human angiogenesis factor in silkworm.The content that mainly comprises two aspects, one is the method for utilizing the Bac-to-Bac baculovirus expression system to make up to contain the recombinant virus of human body interior cutaneous vessel somatomedin, and one is the protein method of utilizing the separation and purification reorganization in the silkworm body that infects of glycogen chromatography column.

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

1, utilize the Bac-to-Bac baculovirus expression system to make up the method for the recombinant virus that contains human body interior cutaneous vessel somatomedin:

Human body interior cutaneous vessel growth factor gene is cloned into the supporting donor plasmid pFastBacHT multiple clone site of Bac-to-Bac baculovirus expression system, donor plasmid is an American I nvitrogene company product, make this gene be positioned at baculovirus polyhedrin body promotor downstream, then this plasmid is transformed into and contains in the genomic competence bacterium of the silkworm baculovirus BmBacmid DH10BmBac cell, contain on the antibiotic culture plate cultivate 13 hours after, obtain positive bacterial plaque by blue hickie screening, subsequently from positive bacterial plaque from the extracting plasmid DNA, this plasmid DNA is the genome of recombinant virus, this DNA is transfected into silkworm cultured cell BmN, isolation medium supernatant liquor after 120 hours, acquisition contains the recombinant virus of human body interior cutaneous vessel growth factor gene.

2, utilize the protein method of glycogen chromatography column separation and purification reorganization in the silkworm body that infects: utilize injection that the above-mentioned recombinant virus of 20 microlitres is injected into the silkworm five-age larva, collect the silkworm sample that infects after 120 hours, add phosphoric acid buffer and homogenate sample then, get supernatant liquor behind the high speed centrifugation, with the raw material of supernatant liquor as extraction recombinant human interior cutaneous vessel growth factor protein, utilize the separation of glycogen affinity column, purifying to obtain corresponding proteins matter, and analyze the lipidated protein of purifying by SDS-PAGE.

The beneficial effect that the present invention has is: utilize silkworm Bac-to-Bac expression system can produce the recombinant baculovirus that contains human body interior cutaneous vessel growth factor gene fast, and in silkworm larva, efficiently express, utilize the glycogen affinity column to realize the sharp separation and the purifying of recombinant protein.

Description of drawings

Accompanying drawing is donor plasmid pFastBacHT figure.

Embodiment

1, research material: silkworm Bac-to-Bac baculovirus expression system is finished by applicant's exploitation, and publishes, (referring to paper: Wu Xiaofeng, Cao Cuiping, Lu Xingmeng.2006, utilize bacterial transposon to make up the Bac-to-Bac rapid gene expression system that is applicable to silkworm.Silkworm industry science, 32 (2): 183-188).Donor plasmid pFastBacHT (Fig. 1) is available from American I nvitrogen company.Various restriction enzymes, ligase enzyme equimolecular biologic operation reagent are available from Japanese Takara company.Human body interior cutaneous vessel growth factor gene is available from U.S. Clontech company.Glycogen affinity column Heparin Sepharose purchases the GE company in the U.S..

2, workflow:

Human body interior cutaneous vessel growth factor gene is cloned between Bac-to-Bac baculovirus expression system supporting the donor plasmid pFastBacHT multiple clone site BamHI and HindIII, make this gene be positioned at baculovirus polyhedrin body promotor downstream, then this plasmid is transformed into and contains in the genomic competence bacterium of the silkworm baculovirus BmBacmid DH10BmBac cell, adding kantlex (concentration 50 micrograms/ML), gentamicin (is cultivated after 13 hours on the culture plate of two kinds of microbiotic of concentration 35 micrograms/ML) and the similar thing X-Gal of galactoside, obtain positive bacterial plaque (hickie by blue hickie screening, be that reorganization has taken place to insert gene), subsequently from positive bacterial plaque from the extracting large plasmid DNA, this plasmid DNA is the genome of recombinant virus, this DNA is transfected in the silkworm single-layer culturing cell BmN, isolation medium supernatant liquor after 120 hours, acquisition contains the recombinant virus of human body interior cutaneous vessel growth factor gene.The above-mentioned recombinant virus of 20 microlitres is inoculated the silkworm five-age larva by the mode of percutaneous injection, observe the incidence of silkworm then, collect after 120 hours by the silkworm of virus infection, the phosphoric acid buffer that adds PH7.2d then with the ratio of 2 milliliters of every boss silkworms, and adopt homogenizer to the meticulous homogenized of sample, get supernatant liquor behind the high speed centrifugation again, with the raw material of supernatant liquor as extraction recombinant human interior cutaneous vessel growth factor protein.Utilize glycogen affinity column Heparin Sepharose separation, purifying to obtain corresponding proteins matter, above-mentioned raw materials added chromatography column makes it and resin-bonded, at last with the NaCl solution one-step elution that contains 1.5M, separate the human body interior cutaneous vessel growth factor protein of reorganization.The sample of purifying utilizes the lipidated protein of SDS-PAGE electrophoretic analysis purifying.

According to actual result, the output of every boss's silkworm larva is estimated about 600 micrograms.

Claims

1, with the method for Bac-to-Bac system expressing human angiogenesis factor in silkworm, it is characterized in that the step of this method is as follows:

1) utilize the Bac-to-Bac baculovirus expression system to make up the method for the recombinant virus that contains human body interior cutaneous vessel growth factor gene:

Human body interior cutaneous vessel growth factor gene is cloned into the supporting donor plasmid pFastBacHT multiple clone site of Bac-to-Bac baculovirus expression system, make this gene be positioned at baculovirus polyhedrin body promotor downstream, then this plasmid is transformed into and contains in the genomic competence bacterium of the silkworm baculovirus BmBacmid DH10BmBac cell, contain on the antibiotic culture plate cultivate 13 hours after, obtain positive bacterial plaque by blue hickie screening, subsequently from positive bacterial plaque from the extracting plasmid DNA, this plasmid DNA is the genome of recombinant virus, this DNA is transfected into silkworm cultured cell BmN, isolation medium supernatant liquor after 120 hours, acquisition contains the recombinant virus of human body interior cutaneous vessel growth factor gene;

2) utilize the protein method of glycogen chromatography column separation and purification reorganization in the silkworm body that infects:

Utilize injection that the above-mentioned recombinant virus of 20 microlitres is injected into the silkworm five-age larva, collect the silkworm sample that infects after 120 hours, add phosphoric acid buffer and homogenate sample then, get supernatant liquor behind the high speed centrifugation, with the raw material of supernatant liquor as extraction recombinant human interior cutaneous vessel growth factor protein, utilize the separation of glycogen affinity column, purifying to obtain corresponding proteins matter, and analyze the lipidated protein of purifying by SDS-PAGE.