Construction And Immunological Effect Evaluation Of Eukaryotic Expression Vector Containing Optimized Codon Of ESAT6from Mycobacterium Tuberculosis

Objective:(1) To construct recombinant expression plasmid pVAX1-ESAT6-Ocontaining codon-optimized ESAT6gene and pVAX1-ESAT6(2) To obtain therecombinant ESAT6protein.(3) To evaluate the immunological effect of the DNAvaccine.Methods:(1) The ESAT6gene was amplified from H37Ra of mycobacteriumtuberculosis DNA geneome by PCR.(2)The ESAT6gene was directed cloned intoexpression plasmid pET42a(+), construct prokaryotic recombinant plasmidpET42a(+)-ESAT6successfully. We express the pET42a(+)-ESAT6fusion protein ofapproximately33kDa in size after induced by IPTG. The SDS-PAGE showed thatthere was specific protein expressed. This protein was identified by Western-Blot.Purified this protein via GST-Bind Resin and Ni-NTA purification system underdenaturing condition and measured the concentration. The purified protein wasexcluded the possible contaminated endotoxin by using polymyxin B agarose,then thesamples was determined the endotoxin activity with Limulus amoebocyte lysate testkit.(3) The ESAT6gene was directed cloned into expression plasmid pVAX1,construct eukaryotic recombinant plasmid pVAX1-ESAT6successfully. Therecombinant was confirmed by PCR and enzyme digestion and gene sequencing.(4)ESAT6coding gene of Mycobacterium tuberculosis was optimized by using the GeneOptimizer software according to the codon usage in mammalian cells. The optimizedESAT6gene was synthesized and inserted into plasmid pVAX1. The recombinant wasconfirmed by PCR and enzyme digestion and gene sequencing.(5)The recombinantplasmids were transfected into Hela cells by means of Sofast Transection Reagent,and investigate transient expression of the fusion protein in eukaryotic cells withRT-PCR and indirect immunofluorescence. Mice were vaccinated intramuascularlywith both wide type and optimized type of pVAX1-ESAT6and the expression was observed by immunohistochemisty assay.(6)BALB/c mice of7weeks old wererandomly divided into4groups. Each mouse was immunized of plasmid DNA byintramuscular injection and electronic pulse, respectively. All mice were boosted atweek2and week4with the same dosage and same method. Flow cytometry (FCM)was used to measure the cells-proliferations of T cells which had been marked withCFSE, to measure the activation of T cells through CD3molecular. And the INF-γreleasing T lymphocytes were enumerated by ELISPOT, Subclass specific antibodiesand the dynamic changes of humoral immune targets were detected by ELISA.Results:(1) Restriction-enzyme digestion and sequencing results showed that theinserted fragment in recombinant plasmid pET42a (+)-ESAT-6was identical toESAT-6gene and was in frame.The pET42a (+)-ESAT6protein was highly expressedin soluble from in E.coli BL21(DE3), and amounted to30.5%of total bacterialproteins. The protein was analyzed good immunoreactivity with mAb by western blot.(2) PCR and enzyme digestion and gene sequencing showed that the insertedfragment in recombinant plasmid pVAX1-ESAT-6was identical to ESAT-6gene andwas in frame.(3) The codon-optimized ESAT6gene was synthesized,The results ofrestriction enzyme digestion and gene sequencing showed that recombinant plasmidhas been constructed successfully.(4)RT-PCR result showed that the recombinantplasmids were successfully expressed in Hela cells. The specific green fluorescencewas observed by immunofluorescence showed that in Hela cells after eukaryotictransfection to express the target protein. In vivo test indicated that the micevaccinated with recombinant plasmids had positive results and showed browngranules in the muscle fibers by immunohistochemisty, but the ESAT6withoptimized codon has stronger brown granules than that with ESAT6.(5)Immunization of both plasmids of ESAT6with optimized codon andpVAX1-ESAT6can induce potent cellular and humoral immune responses in BALB/cin comparison with the negative controls. The the serum interferon-γreleasing and thespleen cell proliferation responses and the number of IFN-γ secretion lymphocyteswere higher than those of control groups (p<0.01), The pVAX1-ESAT6-O groupwas significantly higher than pVAX1-ESAT6(p<0.05), Blank group and the pVAX1 group were no significant differences (P>0.05). The mouse serum anti-ESAT-6IgGantibody level were higher than those of control groups (p<0.05), Anti-ESAT-6IgGantibody level in pVAX1-ESAT6-O group was significantly higher thanpVAX1-ESAT6group (p<0.05).Conclusions:(1)The recombinant prokaryotic expression plasmid pET42a(+)-ESAT6was successfully constructed and the recombinant ESAT6protein was expressed inE.coli and purified.(2)The eukaryotic expression vector pVAX1-ESAT6-O containingcodon-optimized ESAT6gene and pVAX1-ESAT6was successfully constructed, theycould be expressed in vivo and in vitro, and could induce specific cellular andhumoral immune responses. The immumological effect induced by codon-optimizedESAT6DNA vaccine was higher than pVAX1-ESAT6.