Studies On Safety Of Tuberculosis Subunit Vaccine LT70-DPC And Construction Of Fusion Proteins MAR And MRA

Part I Studies on safety of tuberculosis subunit vaccine LT70-DPCObjective: To evaluate safety of tuberculosis fusion protein subunit vaccine which based on ESAT6-Ag85B-MPT64(190-198)-MTB8.4-Rv2626c(LT70)with the complex adjuvant consisting of cationic liposomes dimo-thylidioctyl ammonium bromide(DDA),polyriboinosinic polyribocytidylic acid(poly I:C)and cholesterol(DPC for short).Methods: 1.Acute toxicity test was carried out as follows,mice were immunized with LT70-DPC tuberculosis subunit vaccine and adjuvant DDA at low and high dosages(equivalent to10 000 and 40 000 times of dosage for humans respectively)by subcutaneous and intraperitoneal routes separately,and observed death and bodyweight for toxicity,based on which the maximum tolerated dose(MTD)of the vaccine was calculated.2.Abnormal toxicity test was carried out as follows,mice and guinea pigs were immunized with LT70-DPC tuberculosis subunit vaccine by intraperitoneal route and observed for abnormal reaction,death and bodyweight.3.In systematic allergy test,guinea pigs were immunized with LT70-DPC tuberculosis subunit vaccine,then challenged with the vaccine by intravenous route,and observed for allergic reaction.Results: 1.Acute toxicity test,10 000 times and 40 000 times the normal doses of vaccine and adjuvant DDA injected into mice by subcutaneous and intraperitoneal ways did not cause the abnormal reaction and death of mice.The MTD of mice to LT70-DPC subunit vaccine were: LT70protein>6 600 ?g/kg,DDA>166 600 ?g/kg,Poly I:C>33200 ?g/kg,Cholesterol>50 600 ?g/kg.2.As for the abnormal toxicity test,mice and guinea pigs were all healthy at 7d,and there were no abnormal symptoms,with bodyweight increased.3.There was no allergic reaction found in vaccine groups in systematic allergy test.Conclusion: The results of the acute toxicity test,the abnormal toxicity test and the systematic allergy test showed that there were no side effects detected,which indicated that the LT70-DPC tuberculosis subunit vaccine showed high safety.Part ? Construction of Mycobacterium tuberculosis fusion proteinsObjective: To optimize the expression and purification of the fusion protein ESAT6-Ag85B-MPT64(190-198)-MTB8.4-Rv2626c(LT70)that we have already constructed without any tag.Furthermore,in order to study the expression and purification characteristics of different fusion proteins,the Mycobacterium tuberculosis secretory proteins Ag85 B and MTB10.4,latent antigen Rv1738 were cloned to construct fusion proteins MTB10.4-Ag85B-Rv1738(MAR)and MTB10.4-Rv173-Ag85B(MRA)without His tag.Methods: Following the site-directed mutation were carried out in Ag85 B genes,the 83 th amino acid Isoleucine I-ATA was replaced by Rrginine CGT-R,and the 140 th amino acid Leucine L-TTG was replaced by Lysine AAA-K to attenuated the hydrophobicity of the LT70 protein.Based on molecular cloning techniques,we designed primers with different restriction sites and H37Rv-DNA was used for the PCR template.The antigenic gene was inserted into expression vector p ET-30a(+)by gene engineering.The recombinant plasmid p ET30a-MTB10.4-Ag85BRv1738 and p ET30a-MTB10.4-Rv1738-Ag85 B was constructed in the multiple cloning site.The recombinant plasmid was transformed into E.coli BL-21(DE3)and induced by IPTG.And then the fusion proteins was purified by IEX(ion exchange chromatography)and HIC(hydrophobic interaction chromatography).Results: Comparing mutated LT70 protein with before,the hydrophobicity was still too strong to be eluted from the HIC column.The sequence of the MAR and MRA genes was identified with that in Gen Bank,and the size of fusion proteins were expressed in E.coli BL-21(DE3)in conformity with the expected.The fusion proteins MAR and MRA were expressed in the inclusion body and the supernatant with the molecular weight about 53 KD respectively.Meanwhile,the fusion proteins were purified preliminarily by the IEX and HIC.Conclusion: The optimization of hydrophobicity of LT70 protein was unsatisfactory,remains to be further studied on the optimization.Mycobacterium tuberculosis fusion proteins of MAR and MRA was successfully constructed,and the fusion proteins were purified using different columns.