CN101113460A

CN101113460A - Recombined rhabdovirus AcBac delt CC-GP41 and constructing method thereof

Info

Publication number: CN101113460A
Application number: CNA2007100526290A
Authority: CN
Inventors: 苏正元; 李娟�; 王华林; 邓菲; 胡志红; 袁丽
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2007-07-03
Filing date: 2007-07-03
Publication date: 2008-01-30
Anticipated expiration: 2027-07-03
Also published as: CN101113460B

Abstract

The invention provides a recombinant baculovirus AcBacDeltaCC-GP41 and an establishment method thereof and relates to the technical field of biological engineering technique and pharmacological. The recombinant baculovirus is CCTCC NO.V200702 and the establishment method comprises the steps: the expression system of Bac-to-Bac baculovirus developed by INVITROGEN Company is used, an AcMNPV baculovirus carrier is selected, then double-deletion shuttle carrier Ac-bacDeltaCC is obtained through a reformation of an AcMNPV shuttle carrier and last the recombinant baculovirus AcBacDeltaCC-GP41 is obtained by using insect cell expression system. The invention has the advantages that the virus is the protein recombinant virus which can high effectively express HIV-1 and has the full length of GP41 and the structure and function thereof are close to natural protein and beneficial for studying natural conformation of GP41 and paves the solid foundation for fully explaining mechanism of HIV-1 entering cells and the immune diagnose and vaccine development based on GP41 and the prevention and curing of AIDS.

Description

Recombinant baculovirus AcBac Δ CC-GP41 and construction process thereof

Technical field

The present invention relates to biotechnology and medical technical field, particularly relate to a kind of recombinant baculovirus that can efficiently express HIV-1 envelope glycoprotein GP41.The invention still further relates to the construction process of this recombinant baculovirus.

Background technology

The rhabdovirus expression vector that is most widely used at present is a commercial baculovirus Bac-to-Bac system, and its most frequently used baculovirus vector is an Autographa californica multicapsid nucleopolyhedrosisvirus multinuclear capsid nuclear polyhedrosis virus (AcMNPV).The Bac-to-Bac baculovirus expression system mainly is made up of donor plasmid, shuttle vectors bacmid, insect cell, because this method is quick and easy, the screening cycle of whole recombinant virus is 7～10 days, and can step sizing, simultaneously, donor plasmid can be constructed more restriction enzyme site and transform, and is beneficial to the insertion of foreign gene.

I type human immunodeficiency virus (HIV-1) is the main pathogen that causes China's AIDS (AIDS).Enter in the early stage process of target cell at the HIV-1 infection host, its membrane glycoprotein (GP120 and GP41) plays key effect.The relative gp120 of the variation of gp41 gene fragment is comparatively conservative, wherein GP41 is the coded transmembrane protein of HIV-1 envelope protein encoding gene env, can be divided into film outskirt (ecmdomain), stride film district (transmembrane), 3 parts of cytoplasmic region (cytoplasmic domain), its major function is the fusion of mediation viromembrane and target cell membrane.GP41 has more stable antigenic determinant, and the antibody of generation is sustainable lifelong.Stride the structure of film district, cytoplasmic region and the effect details in fusion is also not fully aware of at present for some mechanism, the especially GP41 of HIV invasion cell, the GP41 native conformation is also understood fully.Clearly also need to carry out more deep research and exploration for these mechanism, and effective expression GP41 full length fragment helps the understanding to the HIV mechanism of causing a disease, develop effective way more effective, more economical, the still less antiviral of side effect, and preparation HIV vaccine.

From both at home and abroad to the research of HIV-1 envelope glycoprotein as can be known, envelope glycoprotein has sufficient development space to the research of HIV-1 medicine.Yet, make its full length fragment be difficult to effective expression because the proteic membrane property of wearing of HIV-1 glycoprotein GP41 causes toxic action to the host.Usually adopt prokaryotic expression methods such as segmentation, low temperature induction at present at GP41.As " expression of HIVgp41 gene segmentation low temperature induction " (Yuan Yuhua etc., " Chinese virusology ", 2004,19 (2), 179-181), improve the expression effect of GP41 albumen in prokaryotic system with the low temperature expression technology exactly.But the product that this article obtains is the prokaryotic expression product, thereby with native protein very big difference is arranged, and is not the expression of full length fragment, and also there are a lot of difficulties in the proteic purifying of GP41 of expressing with this method simultaneously.

International monopoly document mandate publication No. WO2005/010033 is in order to overcome the difficulty that GP41 albumen is difficult for expression on November 8th, 2006 disclosed " the GP41 polypeptide of new solvable and stable trimeric form ", and the polypeptide of the shack between the terminal spiral of the N-that has constructed a kind of modified gp41 ectodomain that has comprised HIV-1 and the C-end spiral, this polypeptide is to have sufficient hydrophilic solubility to stablize tripolymer.But this invention is not the GP41 albumen of total length, exists bigger difference with natural GP41 albumen.

Summary of the invention

The objective of the invention is: recombinant baculovirus AcBac is provided ^{△ CC}-GP41, this recombinant baculovirus can efficiently express HIV glycoprotein GP41, and the GP41 albumen of expression is full-length proteins, and its foreign protein has better antigenic activity, and the eucaryon after expressing has rhetorical function.Another object of the present invention is: recombinant baculovirus AcBac is provided ^{△ CC}The construction process of-GP41, this method are to utilize the exploitation Bac-to-Bac of INVITROGEN company baculovirus expression system, select the AcMNPV baculovirus vector for use, obtain two disappearance shuttle vectors Ac-bac by transforming AcMNPV shuttle vectors (Ac-bacmid) ^{△ CC}, obtain recombinant baculovirus AcBac with the insect cell expression system again ^{△ CC}-GP41.The acquisition of this recombinant baculovirus helps studying the native conformation of GP41, is the mechanism that complete explanation HIV-1 enters cell, also lays a good foundation for the control based on immunodiagnosis, vaccine research and development and the acquired immune deficiency syndrome (AIDS) of GP41 simultaneously.

In order to achieve the above object, the present invention adopts following technical scheme:

Recombinant baculovirus AcBac ^{△ CC}-GP41, by China's typical culture collection center preservation, preservation date: on May 18th, 2007, preserving number: CCTCCNO.V200702.

Recombinant baculovirus AcBac ^{△ CC}The construction process of-GP41 is: with the Bac-to-Bac baculovirus expression system of INVITROGEN company, utilize donor plasmid pFastBacHTc, gp41 is cloned on the multiple clone site of pFastBacHTc with HIV-1 membrane glycoprotein GP41 gene, (6 * his) labels merge frame to make gp41 gene order and 6 Histidines, obtain new donor plasmid pFastBacHTc-gp41, utilize the homology arm on this plasmid that its swivel base is arrived two shuttle vectors Ac-bac that lack again ^{△ CC}On, obtain recombinant shuttle vector Ac-bac ^{△ CC}-gp41, transfection insect cell through cultivating and purifying, can obtain recombinant virus AcBac then ^{△ CC}-Gp41, i.e. CCTCCNO.V200702.

Compared with prior art, the invention has the advantages that: recombinant baculovirus AcBac of the present invention ^{△ CC}-GP41 a kind ofly can efficiently express the proteic recombinant virus of HIV-1 total length GP41, its structure and function are more near native protein, help studying the native conformation of GP41, being the mechanism that complete explanation HIV-1 enters cell, also is that the control of immunodiagnosis, vaccine research and development and acquired immune deficiency syndrome (AIDS) based on GP41 is laid a good foundation simultaneously.

Recombinant baculovirus AcBac ^{△ CC}The construction process of-GP41 has utilized a kind of eukaryotic expression system fast and efficiently---AcMNPV Bac-to-Bac baculovirus expression system, make the structure of recombinant baculovirus simple to operate, quick, safe, efficient, the GP41 albumen of expressing is full-length proteins, and expression amount big, have the back of an expression eucaryon rhetorical function, the foreign protein of expressing has better antigenic activity, greatly facilitates the expression of transmembrane protein and follow-up purifying.

Description of drawings

Fig. 1 is recombinant baculovirus AcBac of the present invention ^{△ CC}The donor plasmid pFastBacHTc synoptic diagram that-Gp41 selects for use.

Fig. 2 is recombinant baculovirus AcBac of the present invention ^{△ CC}The synoptic diagram of-Gp41 structure.

Fig. 3 is recombinant baculovirus AcBac of the present invention ^{△ CC}The synoptic diagram of-Gp41 building process.

Fig. 4 is recombinant baculovirus AcBac of the present invention ^{△ CC}The SDS-PAGE glue of-Gp41 detects figure.

Embodiment

Recombinant baculovirus AcBac ^{△ CC}-GP41, preserving number: CCTCCNO.V200702.

Below in conjunction with accompanying drawing to recombinant baculovirus AcBac of the present invention ^{△ CC}The construction process of-GP41 is described further.

As shown in Figure 3, recombinant baculovirus AcBac ^{△ CC}The construction process of-GP41 follows these steps to order to carry out:

A, from HIV-1 virus, amplify the gp41 gene with polymerase chain reaction (PCR);

B, the explanation of pressing the pGEM-T Easy of U.S. Promega company support agent box, the gp41 gene order of amplification is cloned on the pGEM-T Easy, blue hickie screening and restriction enzyme EcoRI enzyme through sec.-propyl-B-D thiogalactoside and 5-bromo-4-chloro-3-indoles-β-D-galactoside (IPTG/X-gal) are cut evaluation, acquisition contains gp41 gene plasmid pGEM-T-gp41, and carries out sequence verification and do not have sudden change;

C, utilize the donor plasmid pFastBacHTc of INVITROGEN company, Fig. 1 is the synoptic diagram of pFastBacHTc structure, according to the molecular biology operational manual,, obtain the gp41 of band double digestion cohesive end with restriction enzyme BamHI and XhoI double digestion pGEM-T-gp41;

D, will be cloned on the multiple clone site (MCS) with restriction enzyme BamHI and XhoI double digestion donor plasmid pFastBacHTc with the gp41 of double digestion cohesive end, (6 * His) labels merge frame to make gp41 gene order and 6 Histidines, obtain new donor plasmid pFastBacHTc-gp41, the effect of 6 * His label is to be convenient to Ni-NTA resin affinitive layer purification albumen;

E, according to the Bac-to-Bac of INVITROGEN company baculovirus expression system operational manual, new donor plasmid pFastBacHTc-gp41 be transformed into contain Ac-bac ^{△ CC}(number of patent application: in the competent cell of intestinal bacteria DH10B 200710052485.9), by the locus specificity transposition, obtain the chlorampenicol resistant transformant, transformant is cut detection through the PCR detection and the genome enzyme of M13 primer, obtains recombinant shuttle vector Ac-bac ^{△ CC}-gp41;

F, according to the transfection of liposome cell transfecting test kit Lipofectin explanation, with containing Grace ' the s substratum of 10% foetal calf serum with 9 * 10 ⁵Insect cell Sf9 or Sf21 cultivated 3 hours in 27 ℃ in the culture dish of 35mm, the Ac-bac that recombinates ^{△ CC}The DNA transfection of-gp41 is cultivated it based on 27 ℃ of cultivations 5～7 days at the Grace ' s that contains 10% foetal calf serum in insect cell Sf9 or Sf21 then, collects the substratum supernatant, and the proteic recombinant baculovirus AcBac of total length GP41 is expressed in contained being in the supernatant ^{△ CC}-GP41.Fig. 2 is recombinant baculovirus AcBac ^{△ CC}The synoptic diagram of-GP41 structure.

With the recombinant baculovirus AcBac that obtains among the step f ^{△ CC}-Gp41 infected insect cell, collect metainfective insect cell, with the protein expression in the method detection insect cell of SDS-PAGE gel electrophoresis, detected result as shown in Figure 4, obviously the protein band of a visible treaty 41kDa size is consistent with the total length GP41 molecular weight of albumen of expection.Swimming lane shown in the figure contains 3 * 10 approximately ⁵Insect cell, therefore the proteic expression amount of GP41 is about 2 μ g, extrapolates and utilizes the proteic expression amount of this system expression GP41 can reach 50mg/L.

Claims

1. recombinant baculovirus AcBac ^{Δ CC}-GP41 is characterized in that, this recombinant baculovirus AcBac ^{Δ CC}-GP41 is: CCTCC NO.V200702.

2. realize the described recombinant baculovirus AcBac of claim 1 ^{Δ CC}The construction process of-GP41 is characterized in that, this construction process follows these steps to order to carry out:

A, from HIV-1 virus, amplify the gp41 gene with polymerase chain reaction PCR;

B, the explanation of pressing the pGEM-T Easy of U.S. Promega company support agent box, the gp41 gene order of amplification is cloned on the pGEM-T Easy, blue hickie screening and restriction enzyme EcoRI enzyme through IPTG/X-gal are cut evaluation, acquisition contains gp41 gene plasmid pGEM-T-gp41, and carries out sequence verification and do not have sudden change;

C, utilize the donor plasmid pFastBacHTc of INVITROGEN company,,, obtain the gp41 of band double digestion cohesive end with restriction enzyme BamHI and XhoI double digestion pGEM-T-gp41 according to the molecular biology operational manual;

D, will be cloned on the multiple clone site MCS with restriction enzyme BamHI and XhoI double digestion donor plasmid pFastBacHTc with the gp41 of double digestion cohesive end, gp41 gene order and 6 Histidine 6 * His labels are merged frame, obtain new donor plasmid pFastBacHTc-gp41;

E, according to the Bac-to-Bac of INVITROGEN company baculovirus expression system operational manual, new donor plasmid pFastBacHTc-gp41 be transformed into contain Ac-bac ^{Δ CC}The competent cell of intestinal bacteria DH10B in, obtain recombinant shuttle vector Ac-bac ^{Δ CC}-gp41;

F, according to the transfection of liposome cell transfecting test kit Lipofectin explanation, with Grace ' the s substratum that contains 10% foetal calf serum insect cell Sf9 or Sf21 were cultivated 3 hours in 27 ℃ in culture dish, the Ac-bac that recombinates ^{Δ CC}The DNA transfection of-gp41 is cultivated it based on 27 ℃ of cultivations 5～7 days at the Grace ' s that contains 10% foetal calf serum in insect cell then, collects the substratum supernatant, and the proteic recombinant baculovirus AcBac of total length GP41 is expressed in contained being in the supernatant ^{Δ CC}-GP41.