Construction And Application Of Anti-leishmania LPG And Anti-leishmania Glycoprotein ScFv Library

Leishmaniasis,a disease caused by protozoan parasites,widely exists in tropical and subtropical areas such as Asia,Africa,Europe,Latin America,and can be spread by biting of Sandfly that carries Leishmania parasites.There are three major types:cutaneous leishmaniasis,mucocutaneous leishmaniasis and visceral leishmaniasis disease.After being infected by the parasite,human body can build up strongly immunity to prevent further infection.Therefore,it is very convenient for human to study the vaccine which can be used to control spread of the Leishmaniasis.Currently,the research and development of the vaccine are on the way and several vaccines such as inactivated vaccines,genetic engineering of live attenuated vaccines,recombinant vaccines and DNA vaccines,have been used to serve for us.But unfortunately,the vaccine which possesses the broad-spectrum to all of the Leishmania has not come to light.Hence,combination usage of antibiotics is main treatment for the patients at present.Single-chain antibodies are genetically engineered antibodies.In this study,total RNA was extracted from three hybridoma cell lines which can secrete monoclonal antibodies(two of them 3BF(IgG)and WMB(IgG)can bind with Leishmania major glycoprotein;another one bind with Leishmania donovani LPG).Then VH and VL gene fragments were amplified from cDNA of three hybridoma cell lines,and integrated as scFv with a linker.The scFvs were cloned into phagemid vector pCANTAB-5E,and then transformed into Escherichia coli TG1 by heat-shock transformation.To produce phage-display scFvs,the helper phage M13K07 was added to the culture when bacteria were grown up to the log-phase,in which scFv antibody was displayed in N-terminus of the bacteriophage coat protein p?.Then the phage display antibody was applied to two to four rounds of "panning-enrichment-amplification" to screen for specific phage against to glycoprotein or LPG.The specific scFvs were sequenced and their VH gene contained 411bp,390bp,393bp,respectively,and their VL genes contained 359bp,341bp,359bp,respectively.There were four FRs,three CDRs and two characteristic cysteine residues in the VH gene and the VL gene,respectively.Phage supernatants of the positive clones which have accurate sequences,were used to infect E.coli HB2151,and induced by IPTG for soluble antibodies.Further study is the analysis of the physiological,biochemical and functional identification of these proteins.