The Construction And Preparation Of M.tuberculosis Fusion Proteins And The Construction Of PMIG-Bcl6/pMIG-Blimp1

Part Ⅰ The construction and preparation of Mycobacterium tuberculosis fusion proteinsTuberculosis is an ancient chronic infectious disease,while BCG is the only vaccine used for the prevention of tuberculosis,but its effect is limited.The researches on new and effective tuberculosis vaccines were urgently needed.There have been many fusion proteins like H1,H4,H56,M72 stepped into clinical trail.Our lab constructedfusion protein ESAT6-Ag85B-MPT64_190-198-Mtb8.4-Rv2626c（abbreviated as LT70）with good immune protection effect,but the hydrophobicity of which was too strong to be prepared in a large-scale.For a further study,we modified LT70 and constructed pET30a-ESAT6-Mpt64-Mtb8.4-HspX-Rv2626c（abreviated as EMMH26）.We further cloned single Ag85B,and connected it with other latent antigens such as HspX and Rv2626c to construct new fusion proteins,and we did more research on the expression and purification of these proteins.Objective:1)To purify the fusion protein EMMH26 by using different strategies.2)To construct pET30a-Ag85B,pET30a-Ag85B-HspX and pET30a-Ag85B-Rv2626c without His-Tag.3)In order to compare the possible effect of the signal peptide in proteins expression.,we constructed the whole sequence Ag85B（wAg85B,978bp）with the 123bp of signal sequence.Furthermore we constructed pET30a-wAg85B-Rv2626c.4)To investigate the expression and purification of the above fusion proteins.Methods:1)EMMH26 protein stably expressed in E.coli BL21（DE3）.We try to purify it by different strategies:（1）The supernatant was flowing through the weak negative ion DEAE FF column and the hydrophobic column to purify;（2）The supernatant was flowing through the weak negative ion DEAE FF column and molecular sieve superdex 200;（3）The supernatant was salted-out and passed through molecular sieve superdex 200.2)We constructed pET30a-Ag85B,pET30a-Ag85B-HspX and pET30a-Ag85B-Rv2626c by molecular cloning technique withith the linker（GGGGS）₃between the two antigens,which was beneficial to keeping the original protein conformation.We induced the objective proteins expressing in E.coli BL21（DE3）,then optimized the expression conditions,afterwards purified the soluble proteins preliminarily.3)Searched the sequences of wAg85B（978bp）in NCBI genebank and selected the suitable enzyme cut loci to construct the pET30a-wAg85B-Rv2626c.The protein was expressed in E.coli BL21（DE3）,and the expressing condition was optimized.Result:1)Compared with LT70,EMMH26 tend to be expressed in soluable form stablely,and could be purifiedby salt-out preliminarily.Nevertheless,it was difficult for the renaturation of the salt out proteins.We still need further study on the purification of EMMH26.2)We constructed pET30a-Ag85B,pET30a-Ag85B-HspX,pET30a-Ag85B-Rv2626c successfully.The proteins of them could be expressed in E.coli BL21（DE3）respectively.Ag85B and Ag85B-Rv2626c proteins were expressed in supernate while Ag85B-HspX in inclusion body.Negative ion DEAE FF column/hydrophobic column and molecular sieve superdex 75 could be used to purify the Ag85B and Ag85B-Rv2626c proteins.3)We constructed pET30a-wAg85B-Rv2626c successfully,and wAg85B-Rv2626c protein expressed in E.coli BL21（DE3）inclusion body.Compared with Ag85B-Rv2626c protein,we found that Ag85B-Rv2626c without the signal peptide tend to express in soluble form in E.coli BL21（DE3）.Conclusion:Compared with LT70,EMMH26 protein could be expressed more stablely and the hydrophilic was stronger,the salt-out effect was better,but we still need do further improvement on its purification methods.We constructed Ag85B and other three kinds of fusion proteins like Ag85B-HspX,Ag85B-Rv2626c and wAg85B-Rv2626c.Ag85B and Ag85B-Rv2626c are expressed mainly as soluble form.Part Ⅱ The construction of T cell related transcriptional factors Bcl6/Blimp1 on lentivirus vector pMIGBCG is used to prevent tuberculosis,the only vaccine,it has a preventive effect on severe tuberculosis in children,the protection on adult is very low.It is related to BCG induced organism producing specific effect memory T cell(T_EM),rather than the long-term protective central memory T cell(T_CM).Our research focus on how to regulate the generation of long-term effective T_CM increased after subunit vaccine boosted.The differentiation of T cells,whether internal or external factors are mostly ultimately through the role of transcription factors to achieve.Through the bioinformatics analysis,we found many transcription factors that can regulate the development and differentiation of T cells.Further searched the papers on internet,we selected two transcription factors Bcl-6 and Blimp-1 which play a positive and a negative role in regulating memory T cell differentiation respectively.Contructed the recombinant plasmid pMIG-Bcl6 and pMIG-Blimp1,then cotransfect HEK293 T cells with framework plasmid pCl-Eco to package lentivirus.Collecting the virus supernatant to get the transcription factor Bcl6/Blimp1,and the experiments in vivo and in vitro will be done to examine their effects on the differentiation of memory T cells.Objects: Using molecular cloning technique to construct the c DNA of transcription factors Bcl6 and Blimp1 on lentiviral vector pMIG to get pMIG-Bcl6 and pMIG-Blimp1,these might prepare for a further study on transcription factor affected the differentiation of memory T cells and the development of the long-lasting subunit vaccines against tuberculosis.Methods: The c DNA sequences of Bcl6 and Blimp1 were found in the genebank of NCBI.The plasmid p GEM-Bcl6 and p GEM-Blimp1 were purchased from Sino Biological Inc company,the sequence of them in accordance with we searched.We selected the right enzyme cut loci,and constructed Bcl6 and Blimp1 on lentiviral vector pMIG to get the recombinant plasmids pMIG-Bcl6/pMIG-Blimp1 respectively.Results: We used PCR and gene sequencing to verificate the construction of pMIG-Bcl6 and pMIG-Blimp1 were successful.Conclusion: Transcription factors play an important regulatory role on the differentiation of memory T cells.We are trying to use transcriptional factors to regulat T cells differentiation of central memory T cells,thus to improve the immune protection of subunit vaccine.