Construction And Immunological Effect Evaluation Of The DNA Vaccine Containing Codon-optimized CFP10Gene Of Mycobacterium Tuberculosis

Objectives:(1)To construct plasmid pVAX1/CFP10-o containing codon-optimized CFP10gene ofMycobacterium Tuberculosis as a DNA vaccine.(2)To study the immunological effect of the codon-optimized DNA vaccine in mice.Methods:(1) According to the codon bias of Homo sapiens and Mus musculus,weoptimized the codon of the CFP10gene from Mycobacterium Tuberculosis using theGene Optimizer software.The codon-ptimized CFP10gene was synthesized and namedCFP10-o.Then the CFP10-o was inserted into the eukaryotic expression vectorpVAX1in order to construct the recombinant eukaryotic expression plasmidpVAX1/CFP10-o which contained the codon-optimized CFP10gene(CFP10-o).TheCFP10gene was amplified from the genome of Mycobacterium Tuberculosis H37Ra byPCR and inserted into pVAX1to construct recombinant eukaryotic expression plasmidpVAX1/CFP10which contained the wild type CFP10gene.The pVAX1/CFP10-o andpVAX1/CFP10recombinant plasmids were confirmed by sequencing after identificationusing PCR and restriction enzyme digestion.(2)The CFP10gene from the recombinanteukaryotic expression plasmid pVAX1/CFP10was subcloned into the prokaryoticvector pET-42a(+) to construct the recombinant prokaryotic expression plasmidpET42a/CFP-10.The recombinant plasmid pET42a/CFP10was transformed into E.coliBL21(DE3) and the recombinant protein CFP10was expressed with IPTGinduction.The expressed product was purified by Ni+-afinity chromatography andidentified with SDS-PAGE and western blotting.Then the purified recombinant proteinCFP10was removed the endotoxin by ToxinEraserT MEndotoxin Removal kit and testedby TAL (Tachypleus Amebocyte Lysate).(3)The pVAX1/CFP10-o and pVAX1/CFP10plasmids were respectively transfected into Hela cells by Sofast lipofectamine and detected by RT-PCR and indirect immunofluorescence assay(IFA);ThepVAX1/CFP10-o and pVAX1/CFP10plasmids were respectively injected into thequadriceps femoris of the SPF-BALB/c mice using electroporation method.After48h,the expresstion of recombinant protein CFP10in the skeletal muscle of theSPF-BALB/c mice was tested by immunohistochemisty.(4)28SPF-BALB/c mice wererandomly divided into four groups(the pVAX1/CFP10-o group,the pVAX1/CFP10group,the pVAX1group and the NS group) and immunized respectively withpVAX1/CFP10-o, pVAX1/CFP10, pVAX1and NS using electroporation method asthree injection two weeks apart.And then,the CFP10-specific IgG antibody levels in thesera from mice were detected by ELISA at0、2、4、6weeks after immunization. IFN-γlevels in the sera from mice were detected by ELISA at6weeks after immunization. TheCFP10-specific IFN-γ-secreting cells in splenocytes from mice were deteced byELISPOT at6weeks after immunization.The splenocytes from mice were marked withCFSE and stimulated by rCFP10in vitro.Then the levels of T lymphocyte cellsproliferation were detected by Flow cytometry (FCM) and the proliferation Indexs (PI)were analyzed using the flowjo software.Results:(1)The recombinant plasmids of pVAX1/CFP10-o and pVAX-1/CFP10wereconstructed successfully identified by restriction enzyme digestion showing DNA bandsat about300bp and3000bp.And the result of sequencing showed that the inserted genewas consistent with the expected design of the gene sequence.(2)The recombinantplasmid pET42a/CFP10was identified by restriction enzyme digestion and genesequencing which showed the pET42a/CFP10was constructed successfully.The E.coliBL21（DE3）transformed with the plasmid pET42a/CFP10expressed efficiently thesoluble fusion protein CFP10.After a6-h induction with0.1mmol/L IPTG at37℃,theexpression of the37KD protein accounted for about40%of the total bacterial protein.Inaddition,the fusion protein was recognized by CFP10polyclonal antibody.Besides,theprotein was disposed using ToxinEraserT MEndotoxin Removal kit to remove endotoxinwhich was tested by TAL (Tachypleus Amebocyte Lysate).(3)The expected DNA bandswere amplified from the Hela cells transfected with pVAX1/CFP10-o andpVAX1/CFP10plasmids by RT-PCR.The result of IFA showed that specific greenfluorescence were observed both in the Hela cells transfected withpVAX1/CFP10-o and those transfected with pVAX1/CFP10.Furthermore,there wasstronger fluorescenceintensity in the Hela cells transfected with pVAX1/CFP10-o thanthose transfected with pVAX1/CFP10,whereas there was no specific greenfluorescencein Hela cells transfected with pVAX1and NS.More positive brown granules in muscletissue of the mice injected with the pVAX1/CFP10-o plasmid were observed than inmuscle tissue of the mice injected with the pVAX1/CFP10plasmid byimmunohistochemisty. And there were no obvious brown granules in muscle tissue ofthe mice injected with pVAX1and NS.(4) The CFP10-specific IgG antibody levels wason the rise with the times of immunization except the pVAX1group and NS group.At6weeks after immunization,we found that the level of IgG of the pVAX1/CFP10-o groupwas higher than the pVAX1/CFP10group (p<0.05) which was higher than the pVAX1group and NS group (p<0.05) and there was no significantly difference between thepVAX1group and NS group (p<0.05).The level of IFN-γ in serum of pVAX1/CFP10-ogroup was higher than that of the pVAX1/CFP10group and pVAX1and NS group（p<0.01）;And the level of IFN-γ in serum of the pVAX1/CFP10group was higherthan that of the pVAX1and NS group (p<0.01).There was no statistically significancebetween the pVAX1group and the NS group (P>0.05);The splenic T lymphocytes ofmice from each group were cocultured with the purifyed protein CFP10.And the IFN-γreleasing T lymphocytes(Spot Forming Cells,SFC) were enumerated by ELISPOT.TheSFC counts showed higher level in pVAX1/CFP10-o group than pVAX1/CFP10、pVAX1and NS group (p<0.01).And the pVAX1/CFP10group was of higher-level thanpVAX1and NS group (p<0.01).There was no significantly difference between thepVAX1group and NS group (p>0.05);The proliferation Index of the pVAX1/CFP10-ogroup(2.89±0.39%) was distinctly higher than pVAX1/CFP10group(2.06±0.16%)(p<0.05).The proliferation Index of the pVAX1/CFP10group was also higher than thatof pVAX1group and NS group (p<0.05).However, there was no significantly differencebetween the pVAX1group and NS group(p>0.05). Conclusions:(1)The pVAX1/CFP10-o containing the codon-optimized CFP10gene and thepVAX1/CFP10containing wild type CFP10gene plasmids were constructedsuccessfully.(2)The soluble recombinant protein CFP10was prokaryoticly expressed efficiently andthe purified protein was obtained without endotoxin.(3) Hela cells and skeletal muscle of mice transfected with the pVAX1/CFP10-o and thepVAX1/CFP10plasmids could both express the CFP10protein.And the CFP10protein was expressed more effective in Hela cells and skeletal muscle of micetransfected with pVAX1/CFP10-o than that transfected with the pVAX1/CFP10.(4)The codon-optimized DNA vaccine pVAX1/CFP10-o and the wild type DNA vaccinepVAX1/CFP10could both induce CFP10-specific humoral and cellular immuneresponse in BALB/c mice and the pVAX1/CFP10-o could elicit higher-levelCFP10-specific immune responses than the pVAX1/CFP10in BALB/c mice.