摘要
目的
人组织型激肽释放酶基因家族(Kallikreins,KLKs)是近年来新发现的肿瘤标志家族,属于丝氨酸蛋白酶家族,KLK6(Kallikrein6,KLK6)是该家族成员之一,编码人组织型激肽释放酶6(human Kallikrein6,hK6)。成熟的hK6蛋白含223个氨基酸,具有胰蛋白酶样活性,与乳腺癌、卵巢癌、胃癌、肠癌、中枢神经系统疾病密切相关。其研究目的在于构建hK6原核表达载体并制备高效价、高特异度的多克隆抗体和单克隆抗体,为建立检测方法检测组织和体液中hK6奠定基础。
方法
1、提取人乳腺浸润导管癌组织中总RNA,反转录为cDNA,根据GeneBank发表的核苷酸序列D78203,设计KLK6引物,扩增出KLK6基因片段,先克隆入pMD-19T克隆载体SacⅠ、XhoⅠ位点,经菌落PCR、双酶切鉴定和基因测序后确定pMD-19T-KLK6的重组阳性菌,然后提取质粒,胶回收纯化KLK6目的片段,克隆入pET-28b原核表达载体的多克隆位点,转化大肠杆菌BL21,筛选出阳性菌落后,IPTG诱导表达hK6融合蛋白,对诱导时间、温度进行优化并确定蛋白表达形式,扩大培养,大量表达蛋白,进行镍柱纯化、SDS-PAGE及western-blot检测。
2、纯化hK6融合蛋白与弗氏佐剂等体积混合,乳化均匀后,免疫新西兰大白兔和BALB/c小鼠,每10天加强免疫一次,四次免疫后抗体效价达峰值,对新西兰大白兔进行心脏采血,分离出血清,-80℃冰箱保存备用。BALB/c小鼠抗体效价达到10~5后,腹腔加强免疫一次,3天后取小鼠脾脏,制备脾细胞悬液,与预先培养并经8-AG筛选的骨髓瘤细胞SP2/0融合,HAT培养基选择培养,有限稀释法筛选稳定分泌抗体的杂交瘤细胞株,制备腹水获得单克隆抗体。间接ELISA方法检测抗体效价,纯化抗体后检测IgG亚型并进行western-blot检测和免疫组化检测,抗体纯化后保存于-80℃冰箱备用。
结果
1、成功构建pET-28b-KLK6原核表达载体,28℃、1mM IPTG诱导8小时时蛋白表达达峰值,且以可溶性形式表达,其hK6融合蛋白分子量为25KD。
2、镍柱纯化的hK6融合蛋白免疫新西兰大白兔和BALB/c小鼠,得到的抗血清效价为10~7;细胞融合后经有限稀释法筛选出4株分泌抗体的杂交瘤细胞株(DB2C4、DB3D7、BD3G7、DD3B5),回输小鼠腹腔,从腹水中提取单克隆抗体,效价均为51200;IgG亚型分析均为IgG1;western-blot表明,多抗和单抗都能与hK6融合蛋白特异性结合,免疫组织化学分析表明,hK6定位于细胞胞浆,在胃癌、乳腺癌、正常卵巢组织中均有表达。
结论
本课题成功构建了hK6的原核表达载体,通过诱导表达产物制备出高效价、高特异性的抗hK6的多克隆抗体与单克隆抗体,为深入研究hK6功能,及建立高特异性、高敏感性的检测方法,检测组织和体液中的hK6奠定基础。
Objective
The 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.
Methods
1. 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.
Results
1. 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.
Conclutions
hK6 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.
引文
[1] Yousef GM,Luo LY,Scherer SW,et al. Molecular characterization of zyme/proteaseM/neurosin (PRSS9),a hormonally regulated kallikrein-like serine protease[J].Genomics,1999,62:251-259.
[2] Ulf Meyer Hoffert.,Zhihong Wu., Jens Michael Schroder.Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5 Specific Protease Inhibitor[J].PLoS One.,2009,4(2):e4372.
[3] Little SP,Dixon EP,Norris F,et al.A novel and potentially amyloidogenicenzyme cDN- A isolated from Alzheimer’s disease brain[J]. J Biol Chem,1997,272:25135–25142.
[4] Anisowicz A,Sotiropoulou G,Stenman G,et al. A novel protease homolog differentiall- y expressed in breast and ovarian cancer[J].Mol Med.,1996,2(5):624–636.
[5] Yamashiro K,Tsuruoiko N,Kodama S,et al. Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain[J]. Biochim Biophys Act- a ,1997,1350:11–14.
[6] Julie L.V. Shaw,Eleftherios P. Diamandis.Distribution of 15 Human Kallikreins in Tissues and Biological Fluids [J].Clinical Chemistry,2007,53(8):1423–1432.
[7] Diamandis EP,Yousef GM,Soosaipillai AR,et al. Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications[J]. Clin Biochem,2000,33:369–375.
[8] Ghosh MC,Grass L,Soosaipillai A,et al. Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells[J]. Tumour Biol,2004,25:193-199.
[9] Klucky B,Mueller R,Vogt I,et al. Kallikrein 6 induce E-cadherin shedding and promotes cell proliferation, migration, and invasion [J]. Cancer Res,200 7,67(17):8198-8206.
[10] Georgios Pampalakis,Evangelia Prosnikli.A Tumor-Protective Role for Human Kallikrein-Related Peptidase 6 in Breast Cancer Mediated by Inhibition of Epithelial-to-Mesenchymal Transition [J]. Cancer Res,2009,69(9):3779–3787.
[11] Eleftherios P.Diamandis,George M.Yousef,et al.Human Kallikrein 6 (Zyme/Protease M/Neurosin):A New Serum Biomarker of Ovarian Carcinoma [J].Clinical Bio- chemistry,2000,33(7):579–583.
[12] Daniel G. Rosena,Lin Wang,J. Neeley Atkinsonc,et al. Potential markers that complement expression of CA125 in epithelial ovarian cancer[J]. Gynecologic Oncology,2005,(99):267–277.
[13] F Rückert,M Hennig,CD Petraki,et al.Co-expression of KLK6 and KLK10 as prognostic factors for survival in pancreatic ductal adenocarcinoma[J]. British Journal of Cancer,2008,99:1484-1492.
[14] Nagahara H,Mimori K,Utsunomiya T,et al.Clinicopathologic and Biological Signific- ance of Kallikrein 6 Overexpression in Human Gastric Cancer[J].Clin Cancer Re- s,2005,11(19):6800-6806.
[15] Jasmine Singh,Anupam Naran. Expression of kallikrein-related peptidases (KRP /hK5,7,6,8) in subtypes of human lung carcinoma[J]. International Immunopharm- acology,2008,8:300–306.
[16] M Talieri,L Li,Y Zheng,et al.The use of kallikrein-related peptidases as adjuvant prognostic markers in colorectal cancer[J]. British Journal of Cancer,2009,100: 1659–1665.
[17] Yasuo Shinoda,Ken-ichi Kozaki.Association of KLK5 overexpression with invasiv- eness of urinary bladder carcinoma cells Cancer [J]. Japanese Cancer Associa- tion,2007,98(7):1078-1086.
[18] Tadej Strojnik,Rajko Kavala,Diamandis,et al. Prognostic Impact of CD68 and Kallikr- ein 6 in Human Glioma[J] .Anticancer Research,2009(29):3269-3280.
[19] Komatsu N,Saijoh K,Kuk C,et al. Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis:dependence on phenoty- pe,severity and therapy[J].Br J Dermatol,2007,156(5):875-883.
[20]胡成进.Kallikrein6基因在乳腺癌表达、调控及功能研究[D].中国博士学位论文全文数库,2005(7).
[21]张芳,胡成进,陈英剑,孙晓明,郑金锋.山东生物化学与分子生物学会2009年学术会议,2009年.
[22]张芳,陈英剑,胡成进.17-β雌二醇对人卵巢癌细胞株HO8910 KLK6基因表达的调节[J].山东大学学报,2007,45 (1):42-54.
[23]陈奎香,胡成进,陈英剑,等.人组织激肽释放酶6在胃癌中的表达及其临床意义[J].中华检验医学杂志,2009,32(11):1279-1281.
[24]高云,黄宇烽.真核表达系统的研究进展[J].中华男科学,2002,8(4):50-54.
[25] Li Y,Wan L,Yang H,et al. Cloning and recombinant expression of human soluble TRAIL in Pichia pastoris[J]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi.,201 0 ,27(6):1307-1326.
[26]刘高强,章克昌,王晓玲,等.昆虫杆状病毒表达系统的研究与应用进展[J].中国医学工程杂志,2004,24(7):75-79.
[27] Tsumoto K,Yoshimura T. Recombinant proteoliposomes prepared using baculovirus expression systems[J]. Methods Enzymol,2009,465:95-109.
[28]张小霞.外源蛋白表达系统类型的研究进展[J].国外医学卫生学分册,2004,31(4):203-207.
[29] Hockney RC.Recent development in heterogous protein-production in Escherichia coli [J].Trend in Biotechnol,1994,12:456-463.
[30] Jiao J,Zhang ZB,Hu JY,et al. Prokaryotic expression of human vitamin D receptor (hVDR) and its binding activities to Cd and Pb[J].Huan Jing Ke Xue.,2010,31(1 0):2469-2474.
[31]王燕,窦恒利,胡成进.铜绿假单胞菌外膜蛋白OprF原核表达载体的构建及表达[J].山东大学学报(医学版),2010,48 (9):34-39.
[32]李传芬,荆文,胡成进.金黄色葡萄球菌膜蛋白nEBPS原核表达及抗体制备[J].放射免疫学杂志,2010,23(4):411-416.
[33] Kohler G,Mil stein C. Continuous culture of fused cell s secreting antibody of predefined specificity [J] . Nature,1975,256 (5517):495-497.
[34] Lin X,Ma L,Wang J,et al.Preparation of the anti-HLJ1 monoclonal antibodies and establishment of method for detection of the antigen[J].Sheng Wu Gong Cheng Xue Bao. ,2008,24(7):1293-1299.
[35] Liao ZX,Shen JG,Lai XY. Preparation of monoclonal antibodies against human mese- nchymal stem cells[J].Zhejiang Da Xue Xue Bao Yi Xue Bao.,2006,35 (5) :517-522.
[36]王燕,窦恒利,陈世敏,等.抗铜绿假单胞菌外膜蛋白F单克隆抗体制备及夹心ELISA检测方法的建立[J].细胞与分子免疫学杂志,2010,26(9):880-883.
[37]王燕,窦恒利,胡成进.铜绿假单胞菌胶体金免疫层析法的研制和应用[J].标记免疫分析与临床,2010,17(3):174-178.
[38] Matsudaira H,Asakura T,Aoki K,et al.Target chemotherapy of anti-CD147 antibody labeled liposome encapsulated GSH-DXR conjugate on CD147 highly expressed carcinoma cells[J].Int J Oncol.,2010,36(1):77-83.
[39] Constantina D. Petraki,Vassiliki N. Karavana,Pavlos T. Skoufogiannis,et al.The Spectrum of Human Kallikrein 6 (Zyme/ProteaseM/Neurosin)Expression in Human Tissues as Assessed by Immunohistochemistry[J].The Journal of Histochemistry & Cytochemistry Volume,2001,49(11): 1431–1441.
[1] Zhao XL,Chen WQ, Li JM, et al. Preparation of human antibodies to TNF-alpha using risme display technology[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.,2009 ,25 (2):145-149.
[2] Salah Aref, Tarek Sleem, Nadia El Menshawy,et al.Antiplatelet antibodies contribute to thrombocytopenia associated with chronic hepatitis C virus nfection[J].Hmat- ogy,2009,14(5):277-281.
[3] Linlin Qu,Qiurong Li, Haitao Jiang,et al. Effect of Anti-Mouse CD52 Monoclonal Antibody on Mouse Intestinal Intraepithelial Lymphocytes [J].Transplantatio- n,2009,88:766–772.
[4] Swinnen LJ,Costanzo-Nordin MR,Fisher SG,et al. Increased incidence of lymph- oproliferative disorder after immunosuppression with monoclonal antibody OKT3incardiac transplant recipients[J]. N Eng J Med,1990,323:1723-1728.
[5] Opelz G,Dohler B. Lymphomas after solid organ transplantation:A Collaborative Transplant Study report[J]. Am J Transplant,2003,4:222-230.
[6] Shangjin Cui, Changmu Chen, Guangzhi Tong. A simple and rapid immuno- chromatographic strip test for monitoring antibodies to H5 subtype Avian Influenza Virus[J].Journal of Virological Methods, 2008,152:102-105.
[7]佟海侠,王慧涵,张继红,等.白血病免疫表型、细胞遗传学及临床特征的研究[J].中国实验血液学杂志,2009,17(5):1174-1178.
[8] Na Young Ji,Mi Young Park,Yun Hee Kang,et al. Evaluation of annexin II as a potentialserum marker for hepatocellular carcinoma using a developed sandwich ELISA method[J]. International Journal Of Molecular Medicine,2009,24:765-771.
[9] Hong Song,Robert F. Hobbs,Ravy Vajravelu,et al. Radioimmunotherapy of Breast Cancer Metastases withα-Particle Emitter 225Ac:Comparing Efficacy with 213Bi and 90Y[J]. Cancer Research,2009,69(23):8941-8948.
[10] McDevitt MR,Barendswaard E. Anα-particle emitting bismuth-213 labeled antibody (J591) to the external domain of prostate specific membrane antigen[J].Cancer Research,2000,60:95-100.
[11] Song EY,Qu CF,Rizvi SM,et al. Bismuth-213 radioimmunotherapy with C595 anti-MUC monoclonalantibody in an ovarian cancer ascites model[J]. CancerBiol Ther,2008,7:76–80.
[12] Milenic DE,Garmestani K,Brady ED,et al. Targeting of HER2 antigen for the treatm- ent of disseminated peritoneal disease[J]. Clinical Cancer Research,2004,10:7834–78 41.
[13] Desai. D,Brightling .C. Cytokine and anti-cytokine therapy in asthma: ready for the clinic[J]?Clinical and Experimental Immunology,2009,158:10-19.
[14]吴卫星,杨宁,张毓,等.单克隆抗体药物风雨飘摇20年[J].生物技术通讯,2006,17:764-766.