Cloning And Expression Of Leech Derived Tryptase Inhibitor

ObjectiveTo construct the prokaryotic expression vector of LDTI gene,to optimize the condition after the plasmid having been transformed into Escherichia coli[BL21(DE3)]and to express LDTI protein in Escherichia coli[BL21(DE3)],which will lay the first stone for exploring the function and therapeutic action of LDTI gene.Methods1.Gene cloning①Gene amplifying:The LDTI gene was amplified by PCR technique from the template of the plasmid containing LDTI gene and exmined by agarose electrophoresis of 1.4%. Construction of T vector:a 141 bp fragment was obtained and cloned into T vector,the carrier was transformed into Escherichia coli[JM109].Then T vector was extracted from JM109 and exmined by PCR,restricted enzyme,and nucleotide sequencing.③Construction of pET28a-LDTI carrier:After the T vector was digested by restricted enzyme, the target fragment was obtained and connected with another segment of pET28a.The target fragment was subcloned into plasmid pET28a,then the new carrier was transferred into Escherichia coli[JM109]and exmined by PCR,restricted enzyme,and nucleotide sequencing.2.Protein expressingThe recombinant expression plasmid containing LDTI gene was transformed into BL21(DE3).②The target protein expression was induced by isopropyl-B-D-thiogalactopyranoside(IPTG) and exmined by Tricine-SDS-PAGE and Western blot.Then time and consistency of IPTG were adjusted to obtain the most target protein.Results1.A bright 141 bp fragment appeared on the agarose electrophoresis of 1.4%,target gene was ??successfully obtained.2.The new T vector was constructed and examined by PCR,restricted enzyme,and nucleotide sequencing.3.The target carrier pET28a-LDTI was constructed and examined by PCR,restricted enzyme, and nucleotide sequencing.4.The target protein was greatly expressed by pET28a-LDTI and the expression level of LDTI protein was to be approximately 15%after 1 hour's induction in the concentration of 0.7mmol/L IPTG at 33℃.ConclusionPET28a-LDTI was successfully constructed and could highly express objective protein in BL21 (DE3),which would further help in anlysising the funtion of LDTI.