CN101721693B - Improved recombinant bacillus calmetter Guerin (BCG) - Google Patents

Abstract

The invention discloses an improved recombinant bacillus calmetter Guerin (BCG), belonging to the technical field of new medicaments. The immune protecting effect of a unique anti-tuberculosis vaccine BCG for tuberculosis is not exact and the tuberculosis morbidity gradually rises over the past 10 years. Therefore, the development of a more effective anti-tuberculosis vaccine is important. Gene sequences of mycobacterium tuberculosis ESAT6 and Ag85A are inserted into shuttle plasmids of colon bacillus-mycobacterium tuberculosis for forming recombinant plasmids; and the recombinant plasmids are converted into the BCG to form a recombinant anti-tuberculosis vaccine. Proved by research, the recombinant BCG is used for expressing introduced foreign genes and is a novel anti-tuberculosis vaccine.

Description

A kind of recombinant bacillus Calmette-Guerin vaccine of improvement

Technical field

The invention belongs to biological and new medicine technology (medical biotechnology) field.

Background technology

(bacillus calmette querin be prevention unique vaccine lungy BCG), but its immune protective effect is unstable, is 0-80% to the preventive effect of adult's tuberculosis for bacillus calmette-guerin vaccine.The various countries scholar just is being devoted to the research of more efficient, safe tuberculosis vaccine.Exogenous gene is introduced BCG, and to make foreign DNA continual and steady expression in BCG being recombinant BCG, is the focus of developing novel tuberculosis vaccine at present.The character that depends on to the exogenous gene of its introducing whether satisfactory for result of recombinant BCG, and the expression efficiency of foreign DNA.Big quantity research shows, (Early secretory antigen target 6 ESAT6) is the main secretion antigen of tubercule bacillus, can induce stronger immunne response for tubercule bacillus immune protective antigen Ag85A and early stage secreted target antigen 6.Adopt independent ESAT6 or Ag85A recombinant BCG, find its immanoprotection action behind the immune animal not as good as BCG, its reason possibly be that its antigenic stimulus is not enough, possibly be that the expression efficiency of its carrier is lower in addition on the one hand.Shuttle expression plasmid pMV361 has the promoter of the replication origin and the MTB HSP60 of kalamycin resistance gene, bacillus coli-mycobacteria.In addition, it sticks site attP and integrator gene int in addition, whereby, can directionally be incorporated on the genomic chromosome attB of BCG, thereby be different from general shuttle vector, thereby express the destination protein that inserts BCG more accurately, enduringly.This research adopts mycobacterium tuberculosis Ag85A and ESAT6 for introducing gene, is carrier with the shuttle plasmid pMV361 of accurate stably express, construction recombination plasmid, and this plasmid imported BCG, make up recombinant BCG.

Summary of the invention

1. the structure of recombiant plasmid pMV361-Ag85A-ESAT6 and evaluation

According to the design of the gene order reported among the Genbank and Synthetic 2 to primer; With tubercule bacillus H37RV genomic DNA (tubercule bacillus H37RV genomic DNA is preserved by this chamber) is template; Polymerase chain reaction (Polymerase ChainReaction, PCR) amplification mycobacterium tuberculosis Ag85A and early stage secretion target antigen 6 (ESAT6) gene.Introduce Mun I and NspV restriction enzyme site to linker, the two ends of Ag85A downstream and 45 bases of ESAT6 upper reaches introducing respectively; Through SOE method (overlap extension; Gene splicing by overlap extension); Amplification Ag85A-ESAT6 fusion gene, glue reclaims test kit and reclaims genes of interest.Plasmid extraction kit extracts pMV361 empty plasmid (the pMV361 empty plasmid is so kind as to give by professor Xu Heng of Sichuan University, and this chamber is conventional preserves), and agarose gel electrophoresis shows that the plasmid extraction effect is good.Restriction endonuclease Mun I and NspV be double digestion fusion gene Ag85A-ESAT6 and pMV361 empty plasmid respectively, and glue reclaims test kit and reclaims genes of interest behind the enzyme action.Be connected with 8: 1 the ratio of mol ratio of pMV361 empty plasmid in fusion gene Ag85A-ESAT6, connect connect 4h under 16 ℃ of conditions of test kit after, direct transformed competence colibacillus bacillus coli DH 5 alpha.The picking positive colony extracts plasmid, adopts Mun I and NspV double digestion, pcr amplification to identify respectively, identifies that correct plasmid send the evaluation of checking order of Invitrogen company., above-mentioned 3 kinds of methods confirm this construction of recombinant plasmid success after identifying, and called after pMV361-Ag85A-ESAT6.

2. recombinant bacillus Calmette-Guerin vaccine rBCG-Ag85A-ESAT6 makes up and identifies

Bacillus calmette-guerin vaccine changes kind of a cultivation preparation competent cell, and the pMV361-Ag85A-ESAT6 plasmid purification that above-mentioned structure is successful is experienced BCG cell, called after rBCG-Ag85A-ESAT6 after electroporation transforms.Behind the electroporation discharge off, get bacterium liquid and coat on the flat board that contains kanamycin, 37 ℃ left standstill for 3～4 weeks, and single bacterium is inoculated in fluid medium continuation antibiotic-screening on the picking flat board then.Treat that Mycoderma grows, bacterial genomes is extracted test kit and is extracted the recombinant bacillus Calmette-Guerin vaccine genome, is that template is carried out the PCR evaluation with the recombinant bacillus Calmette-Guerin vaccine genome.Identify that through PCR successful genome send the order-checking of Invitrogen company not undergo mutation with the genes of interest of proof importing bacillus calmette-guerin vaccine or disappearance etc.Meanwhile, same procedure imports BCG as contrast with the pMV361 empty plasmid, called after rBCG-361.

3. abduction delivering and the evaluation of genes of interest in recombinant bacillus Calmette-Guerin vaccine

Through antibiotic-screening, PCR identify and the gene order-checking proof make up successful recombinant bacillus Calmette-Guerin vaccine leave standstill cultivate treat that Mycoderma grows several weeks after, thermal induction 1h, collection culture fluid supernatant, the bag filter of packing into is concentrated into original volume 1/10.With the thalline carrying out ultrasonic bacteria breaking behind the abduction delivering, collect broken bacterium supernatant and deposition.Adopting monoclonal antibody that these 3 kinds of gleanings are carried out Western-blotting and identify whether express genes of interest and expression thereof with the proof recombinant bacillus Calmette-Guerin vaccine, is contrast with BCG and rBCG-361 in the research.

Advantage of the present invention is: mycobacterium tuberculosis immune protective antigen Ag85A and early stage secreted target antigen 6 are the main secretion antigen of tubercule bacillus; Wherein ESAT6 only is present in the mycobacterium tuberculosis toxic strain and the gene protein that lacks among the BCG; Big quantity research shows that the two all can induce body to produce stronger immunne response, and immunotoxicity a little less than.PMV361 have MTB HSP60 promoter, stick site attP and integrator gene int; Whereby; Can directionally be incorporated on the genomic chromosome attB of BCG, thereby be different from general shuttle vector, thereby express the destination protein that inserts BCG more accurately, enduringly.Is that carrier imports BCG with Ag85A and ESAT6 fusion gene through pMV361; Make exogenous gene in the continual and steady expression of BCG; The Ag85A that utilizes plasmid pMV361 expression system to efficiently express can solve because of the BCG expressing quantity low; And to the insufficient difficult problem of body immune system immunostimulation, the efficiently expressing of ESAT6 then solved BCG because of lack this gene can not stimulating immune system to the ability of anti-mycobacterium tuberculosis.Through above-mentioned 2 kinds of proteic continuous stimulations, can induce body to produce specific immune response, thereby reach the purpose of anti-mycobacterium tuberculosis infection.In sum, this gene recombinaton BCG can produce stronger, the safer immune effect efficiently than BCG.

Description of drawings

Accompanying drawing 1 explanation:

1:Ag85A-linker PCR product (1068bps);

2:Linker-ESAT-6 PCR product (338bps);

3:Ag85A-linker-ESAT-6 fusion gene (1362bps);

4:pMV361 empty plasmid (4445bps);

5:rpMV361-Ag85A-linker-ESAT-6 recombiant plasmid (5791bps);

6: the recombiant plasmid double digestion is identified;

The 7:pMV361 empty plasmid contrasts with the condition double digestion;

8: recombiant plasmid PCR identifies, can amplify genes of interest;

The 9:pMV361 empty plasmid is not seen amplified band with the contrast of the PCR under the condition.

Accompanying drawing 2:rBCG-Ag85A-ESAT6 genome extracts and PCR identifies

The 1:rBCG-Ag85A-ESAT6 genome;

2:rBCG-Ag85A-ESAT6 genome PCR identifies;

3:rBCG-361 empty plasmid genome;

PCR identifies under 4:rBCG-361 empty plasmid genome the same terms, does not see amplified band;

The 5:BCG genome;

PCR identifies under 6:BCG genome the same terms, does not see amplified band;

Proteic expression of accompanying drawing 3:rBCG-Ag85A-ESAT6 and Western-blotting identify

The specific embodiment:

1. primer design is with synthetic

Design following 2 pairs of primers according to the gene order of reporting among the Genbank: P1:5 ' GC C AAT TGT AAT GCA GCTTGT TGA CAG GGT T-3 '; P2:5 '- GCT TCC ACC TCC TCC GCT TCC ACC ACC TCC GCT TCC ACC GCC ACCGGC GCC CTG GGG CGC GGG-3 '; P3:5 '- GGT GGC GGT GGA AGC GGA GGT GGT GGA AGC GGA GGA GGT GGA AGCACA GAG CAG CAG TGG AAT-3 '; P4:5 '-GC T TCG AAT CTA TGC GAA CAT CCC AGT GAC-3 '.Underscore partly is respectively Mun I and Nsp V restriction enzyme site among P1 and the P4, and underscore partly is the linker of complementary 45 bases among P2 and the P3.By Shanghai Invitrogen company synthesizing and purifying.

2. the pcr amplification of genes of interest

The pcr amplification condition is: 94 ℃ of preparatory degeneration 5min, and 94 ℃ of degeneration 45s, 62 ℃ of renaturation 1min, 72 ℃ are extended 1min, circulate altogether 30 times, and last 72 ℃ are continued to extend 10min.With the H37RV genomic DNA is template, is primer with P1, P2 at first, and amplification Ag85A-linker product is a primer with P3, P4, amplification linker-ESAT6 product.Being primer with P1, P4 then, is template with above-mentioned 2 kinds of PCR products, i.e. amplification obtains the Ag85A-linker-ESAT6 fusion gene.

3. the extraction of DNA

After the E.coliDH5 α bacterium liquid that will contain the pMV361 plasmid increases bacterium in a small amount; Adopt the plasmid extraction kit of Omega company to extract plasmid; The operation by specification carries out, and with appropriate amount of buffer solution or distilled water dissolving, the plasmid that takes a morsel detects its purity and concentration through agarose gel electrophoresis at last.

4. the structure of recombiant plasmid pMV361-Ag85A-ESAT6 and evaluation

Plasmid extraction kit extracts the pMV361 empty plasmid, and agarose gel electrophoresis shows that the plasmid extraction effect is good.Respectively pMV361 empty plasmid and Ag85A-linker-ESAT6 fusion gene are carried out Mun I and NspV double digestion, 37 ℃ of enzyme action 4h, glue reclaims test kit and reclaims the purification genes of interest.Be connected with 8: 1 the ratio of mol ratio of pMV361 empty plasmid in fusion gene Ag85A-linker-ESAT6; Direct transformed competence colibacillus bacillus coli DH 5 alpha behind the connection test kit connection 4h; After cultivating increment, 37 ℃ of concussions of picking positive colony extract plasmid; Adopt Mun I and NspV double digestion, pcr amplification to identify respectively; Enzyme action and pcr amplification identify that all correct plasmid send the evaluation of checking order of Invitrogen company, and above-mentioned 3 kinds of methods are identified all correct recombiant plasmid called after pMV361-Ag85A-ESAT6.

5. recombinant bacillus Calmette-Guerin vaccine rBCG-Ag85A-ESAT6 makes up and identifies

5.1BCG commentaries on classics kind and cultivation

The conventional BCG that cultivates of this laboratory is changeed kind in the Sauton culture medium, and 37 ℃ leave standstill to cultivate and treat that Mycoderma grows 4～5 weeks, and thalline is white in color or displaing yellow slightly, when having gauffer to cover whole cultivation liquid level, promptly can be used for the preparation of BCG competent cell.

5.2 the extraction of recombinant plasmid dna

The single bacterium of picking recombiant plasmid pMV361-Ag85A-ESAT6 is in LB fluid medium (containing the 20ug/ml kanamycin), and 37 ℃ of joltings are spent the night, and it is subsequent use to extract plasmid.

5.3 the preparation of competence BCG cell

Be taken at well-grown 3 all left and right sides cultures on the Sauton culture medium; To wherein dripping final concentration is 4% glycine, and 37 ℃ are continued to leave standstill and cultivate 24h, take out culture bottle; Add the sterilization bead; Place in the shaken cultivation case, thermal agitation is cultivated 4h under the 180r/min condition, fully smashes Mycoderma and makes it to become uniform bacteria suspension.With the centrifugal collection thalline of bacteria suspension, bacterial precipitation is with sterilization 10% glycerol washing 3 times after the taking-up, and the last resuspended thalline of reuse 10% glycerol promptly obtains the BCG competent cell.

5.4 the preparation of electroporation cup

The electroporation cup is immersed in 75% ethanol always before, and electroporation took out the same day, behind the drip-dry ethanol, tilts to put into superclean bench, opens uviol lamp and air blast 5h, lets remaining ethanol thoroughly volatilize.

5.5 the electroporation of recombiant plasmid transforms

Respectively with the pMV361-Ag85A-ESAT6 plasmid of purification cell, recombinant BCG called after rBCG-Ag85A-ESAT6 through the electroporation transformed competence colibacillus.Concrete grammar is following: get competent cell 400ul and add the 10ul plasmid, pressure-vaccum is mixed repeatedly changes in the 0.2cm electroporation cup after evenly with the application of sample rifle, at voltage 6.25kv/cm, and electric capacity 2.5C, discharge is 2 times under the resistance 720 Europe conditions, discharges for 2 times to be spaced apart 5min.Ice bath 10min immediately behind the discharge off adds an amount of Sauton culture medium, and 37 ℃ of joltings are spent the night.Get bacterium 200ul coating and contain on the Sauton flat board of 20ug/ml kanamycin, 37 ℃ leave standstill 4 weeks of cultivation.

5.6 the PCR of recombinant BCG identifies and gene order-checking is identified

Single bacterium colony on the picking Sauton flat board; Be inoculated in the culture fluid that contains the 20ug/ml kanamycin; 37 ℃ leave standstill to cultivate and carry out the antibiotic resistance screening several weeks, treat that Mycoderma covers with the culture fluid surface, collect the reorganization thalline; Bacterial genomes is extracted test kit and is extracted genome PCR and identify, PCR identifies that successful genome send the evaluation of checking order of order-checking company.

6. genes of interest is identified at the abduction delivering and the Western-blotting of recombinant bacillus Calmette-Guerin vaccine kind

6.1 the abduction delivering of recombinant bacillus Calmette-Guerin vaccine and the preparation of expression product

The recombinant bacillus Calmette-Guerin vaccine that contains exogenous gene leaves standstill in Sauton culture fluid (containing the 20ug/ml kanamycin) to be cultivated for two weeks, put 45 ℃ of water-bath thermal induction 1h rapidly, the centrifugal 10min collection of 6000r/min bacterium.It is subsequent use to collect the culture fluid supernatant.The ice-cold PBS liquid of thalline (Ph7.2) washing 2 times, centrifugal collection thalline, PBS is resuspended; Carrying out ultrasonic bacteria breaking under the ice bath: supersonic frequency 20KHz, ultrasonic 10s is 10s intermittently, altogether ultrasonic 30min; 4 ℃ afterwards, the centrifugal 10min of 1200r/min get brokenly the bacterium supernatant and precipitate subsequent use.Change culture fluid over to bag filter (minimum molecular cut off is 8.8kDa) with the micropore filter filtration sterilization, it is subsequent use that PEG6000 is concentrated into 1/10 ,-20 ℃ of preservations of original volume.

6.2 the Western-blotting of destination protein identifies

Get above-mentioned albumen sample respectively, add equal-volume 2 * SDS sample-loading buffer (100mMpH6.8Tris-CL, 100mM beta-mercaptoethanol, 4%SDS; 0.2 bromophenol blue, 20% glycerol), fully boiling water boils 5min behind the mixing; Centrifugally go up appearance, carry out the SDS-PAGE electrophoresis, first 80v voltage stabilizing electrophoresis to the separation gel interface; Voltage is added to 120V to the electrophoresis end, cut the purpose adhesive tape and adopt half-dried transfer system that above-mentioned protein transduction is printed on the pvdf membrane, commentaries on classics film condition is: 10V, 70min.Change film and finish back 5% defatted milk powder sealing 1h; TBST buffer washing 5min * 3 time, it is anti-to add mouse anti ESAT6 (TBS liquid dilution 1: 600) IgG one, and 4 ℃ are spent the night; Next day, rewarming was placed 1h to room temperature; TBST washing 5min * 3 time add goat anti-mouse igg two anti-(TBS dilution 1: 1800) room temperature 1h of HRP labelling, DAB colour reagent box colour developing behind TBST washing 5min * 3 time.Digital camera is taken a picture and is deposited at the end.

Claims (3)

1. gene recombinaton bacillus calmette-guerin vaccine; It is characterized in that adopting escherichia coli-mycobacterium shuttle plasmid pMV361 is carrier; The applying gene recombinant technique carries out recombination to construct recombiant plasmid pMV361-Ag85A-ESAT6 with Mycobacterium tuberculosis Ag85A and ESAT6 gene and carrier pMV361; And this recombiant plasmid imported the bacillus calmette-guerin vaccine genome with the electroporation mode; Identify through abduction delivering in recombinant bacillus Calmette-Guerin vaccine and Western-blotting, confirm that genes of interest at recombinant bacillus Calmette-Guerin vaccine genome stable existence and highly effective expressing recombinant protein, obtains novel tuberculosis vaccine rBCG-Ag85A-ESAT6.

2. the purposes of the described recombiant plasmid pMV361-Ag85A-ESAT6 of claim 1 can be applicable to the structure of recombinant bacillus Calmette-Guerin vaccine, also is the carrier of mycobacterium tuberculosis Ag85A and ESAT6 gene simultaneously.

3. the construction method of the said recombiant plasmid pMV361-Ag85A-ESAT6 of claim 1 after the Linker base sequence of introducing 45bp between Gene A g85A and ESAT6 forms fusion gene, carries out the recombination to construct recombiant plasmid with itself and pMV361 again.

Images