Cloning, Expression Of Granulysin CDNA From Human And Granulysin's Function Study

Objective To clone cDNA of granulysin,construct prokaryotic expression vector carrying granulysin and express granulysin in E.coli.To purify GST-granulysin fusion protein from E.coli and identify its antibacterial activity in vitro.Methods Human PBMC were stimulated with Mtb-Ag and incubated in IL-2 containing media for 11 days to generate MtbAT.Total RNA was extracted and granulysin cDNA was obtained by RT-PCR from MtbAT.PCR product was collected from electrophoresis gel and cloned into clone vector pCDNAâ…¡,constructing the recombination vector,pCDNAâ…¡-granulysin.Positive clones were collected and sequenced after identification by restricted enzyme digestion.Correct recombination vector pCDNAâ…¡-granulysin confirmed by sequencing was sub-cloned into prokaryotic expression vector pGEX-4T1 to construct recombinant expression vector pGEX-4T1-granulysin.Positive clones were screened and identified by restriction digestion,pGEX-4T1-granulysin was transformed into E.coli BL21. Expression of GST- granulysin fusion protein in E.coli BL21 was induced by using IPTG,and identified by SDS-PAGE.The transformant E.coli was sonicated following induction by IPTG.The soluble extract containing GST-granulysin fusion protein was separated and purified by affinity chromatography with glutathione agarose. Bactericidal activity of the purified protein was analyzed by MTT assay in E.coli BL21, Staphylococcus aureus Cowanâ… ,alpha hemolytic Streptococcus S34Sr and Eberthella typhosa 0901.Results1.A considerable amount of MtbAT was obtained from PBMC stimulated by Mtb-Ag,among which the percentage ofγδT cell was 77.62%.438 bp targeted fragment was successfully amplified from total RNA extracted from MtbAT cells.2.Sequencing analysis showed that the cloned eDNA sequence was consistent with that in Genbank,except a genetic code ACC mutationto ATC,resulting in threonine 119 mutation to isoleucine.3.E.coli BL₂₁ carrying vector pGEX-4T1-granulysin expressed the GST-granulysin fusion protein after induced by IPTG,which displayed a band with relative molecular mass(Mr) of 44 kDa on SDS-PAGE.After affinity chromatography,we obtained a single protein band at about molecular weight of 44 kDa.4.The result of bactericidal activity analysis showed a potential inhibition to the growth of some species of pathogenic organisms.The bacteriostasis rates of purified product against Staphylococcus aureus and S.sanguis were 75.47%and 86.75% respectively,contrasting to 38.63%and 30.91%in Salmonella enterica and E.coli BL21 respectively.Conclusions Clone vector and prokaryotic expression vector of granulysin were successfully constructed.GST-granulysin fusion protein was expressed and purified, which had highly effective bactericidal activity against Gram positive bacteria.