Construction Of The Prokaryotic Expression Vecter Of Human Kallikrein 6 And Preparation Of It's Antibodies

ObjectiveThe kallikrein gene family (KLKs) is one lately discovered tumor-marker family, belong to the secreted serine protease families. The human kallikrein gene 6 (KLK6), which encodes for human kallikrein 6 protein (hK6), is one member of the gene family. The mature hK6 consists of 223 amino acids with trypsin-like activity. There is a close relationship beween hK6 and breast cancer, ovarian cancer, stomach cancer, colon cancer, central nervous system diseases. The research is to construct the prokaryotic expression vector pET-28b-KLK6 and preparate polyclonal antibody and monoclonal antibody which show high titer and good specificity.It is the base to establish different assays for the detection of hK6 in human tissues and fliuds.Methods1. A pair of primers were synthesized according to D78203 of GeneBank. Total RNA was extracted from human invasive ducal breast cancer tissues.The KLK6 fragment was amplified from cDNAs by PCR and cloned into SacⅠand XhoⅠsites of a cloning vector pMD19-T. By PCR,restriction endonuclease digestion analysis and DNA sequencing,the recombinant plasmid pMD19-T-KLK6 was confirmed. The KLK6 fragment was extracted by DNA Gel Extraction Kit to construct the prokaryotic expression vector pET-28b-KLK6,which was transformed into E.coli BL21. With IPTG induction, the antigen hK6 was produced.To search the best conditions for protein expression,we chose different temperatures and times firstly, and detected in which fraction the hK6 fushion protein was mainly present of the cell lysate. At last the expressed protein was analyzed by SDS-PAGE and Western-blot, purified by Ni-NTA Spin Kit.2. The hK6 fusion protein was applied for immunization of New Zealand white rabbits after mixing with Freund's Adjuvant. It's antiserum was collected and conserved at -80℃. Applied Western blot with the purified polyclonal antiserum hK6 and validated the antibody specific for hK6 protein. In addition, also used immunocytochemical staining to detect the hK6 fusion protein expression in normal or cancer tissues. A BALB/c mice was immunized with the purified hK6 fusion protein. When the titre reached 1:100000, the immunized mice were boosted by intraperitoneal injection with hK6 in PBS without adjuvant. Three days later, the spleen was dissected and the spleen cells were fused with SP2/0 cells. Fused cells were cultured and selected in HAT medium. The hybridoma cell lines secreting monoclonal antibodies were screened with limiting dilution assay and ELISA assay. Monoclonal antibodies were extracted from the ascites .And it was identified by indirect ELISA assay and western-blot and purified by protein A agarose.Immunocytochemical staining was applied for hK6 expression in the tissues of gaster cancer , normal breast and ovary tissues.Results1. The prokaryotic expression vector pET-28b-KLK6 was successfully constructed and hK6 fusion protein was expressed induced IPTG.Detected the hK6 protein expression by SDS-PAGE and western-blot,the peak appeared at 28℃and the 8^th hour induction of 1mM IPTG. SDS-PAGE analysis showed a size of protein band of 25KD,which was mainly present in the soluble fraction of the cell lysate.2. Polyclonal antibodies were obtained after four booster injections.Their antibody titres in serum were 1:10000000. The hybridoma cell lines secreting monoclonal antibodies were screened with limiting dilution assay. 4 hybridoma cell strains were selected,named DB2C4,DB3D7,BD3G7 and DD3B5. Monoclonal antibodies were extracted from the ascites. The monoclonal antibodies with titer 51200 were obtained after large quantity preparation,detected by indirect ELISA assay. Isotyping showed that all four clones produced IgG1 antibodies. Western-blot confirmed the polyclonal antibody and monoclonal antibody against hK6 were specific for hK6 fusion protein and reacted with a 25,000 dalton molecule. To further confirm antibodies specificity, we performed immunocytochemistry staing.hK6 was mainly expressed in cytoplasm.The positive reaction was found in tissues of gaster cancer or breast cancer or normal ovary by immunocytochemical staining.ConclutionshK6 was successfully expressed in the prokaryotic expression vector and polyclonal antibody and monoclonal antibody was produced which show high titer and good specificity. It provided the basis for further research into functions of hK6 and into detecting hK6 in human tissue and fluid.