THE CONSTRUCTION OF HPV16 L1 RECOMBINANT PLASMID AND ITS EXPRESSION IN MAMMALIAN CELLS

Ｐｒｅｖｉｏｕｓｓｔｕｄｉｅｓｈａｖｅｄｅｍｏｎｓｔｒａｔｅｄｔｈａｔｇｅｎｉｔａｌｉｎｆｅｃｔｉｏｎｗｉｔｈｈｉｇｈ－ｒｉｓｋｔｙｐｅｓｏｆｈｕｍａｎｐａｐｉｌｌｏｍａｖｉｒｕｓ（ＨＰＶ）,ｍｏｓｔｏｆｔｅｎＨＰＶ１６,ａｒｅｔｈｅｍｏｓｔｓｉｇｎｉｆｉｃａｎｔｒｉｓｋｆａｃｔｏｒｆｏｒｔｈｅｄｅｖｅｌｏｐｍｅｎｔｏｆｃｅｒｖｉｃａｌｃａｎｃｅｒ．ＨＰＶ１６ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎｓａｎｄｎａｔｉｖｅｖｉｒｉｏｎｓｏｎｌｙｅｘｉｓｔｉｎｄｉｆｆｅｒｅｎｔｉａｔｅｄｓｕｐｅｒｆｉｃｉａ…     

HPV-6/11 L1/E6、HPV-6/11 L1/E7嵌合DNA疫苗质粒的构建

目的　构建HPV 6 / 11L1/E6、HPV 6 / 11L1/E7预防、治疗性DNA疫苗质粒。方法　运用PCR扩增HPV 6 /11L1、E6、E7序列 ,PCR产物连接至pGEMT Easy质粒 ,测序鉴定正确后 ,分别连接至真核表达载体pVAX ,再用酶切、连接而构建成HPV 6 / 11L1 E6 /E7 pVAX质粒。运用电穿孔法将所获得质粒转染COS 7细胞 ,免疫组化检测蛋白表达情况。结果　所构建质粒的插入目的片段测序正确 ;免疫组化检测在胞浆胞核可见棕黄色点状阳性产物沉积。结论　所构建的HPV 6 / 11L1 E6 /E7 pVAX融合蛋白表达质粒可在体外表达L1 E6 /L1 E7蛋白 ,为今后进行DNA疫苗的动物实验及临床实验研究做好准备     

宫颈癌患者人乳头瘤病毒16型E6基因的克隆及序列分析

目的　分析中国陕西地区宫颈癌患者人乳头瘤病毒 16型 (HPV16型 )E6基因的结构特点。方法　采用PCR技术扩增了 1例HPV16阳性宫颈癌患者癌组织中HPV16E6基因 ,并对其进行了克隆及全序列分析。结果　成功地克隆了HPV16E6基因 ,与GenBank收录的原型相比 ,E6基因中有 2个碱基发生突变 ,推导其编码的氨基酸有 1个发生了变化。结论　中国人宫颈癌HPV16E6基因结构有一定的变异     

DETECTION OF HPV 16 E_6G ENE IN CERVICAL CANCER TISSUE

ＤＥＴＥＣＴＩＯＮＯＦＨＰＶ１６Ｅ_６ＧＥＮＥＩＮＣＥＲＶＩＣＡＬＣＡＮＣＥＲＴＩＳＳＵＥ￥ＹｕａｎＹｕｈａｎｇ（袁育康）；ＬｉｕＨａｎｘｉｎ（刘汉馨）；ＬｉｕＺｂｅｎｇｒｅｎｇ（刘正风）；ＣｈｕＹｏｎｇｌｉｅ（楚雍烈）（ＤｅｐａｒｔｍｅｎｔｏｆＭｉ?..     

PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16L1／E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.     

女性外阴病变及其与HPV16/18、HPV6的相关性

目的研究外阴病变及其与HPV16/18、HPV6的关系。方法收集西安交通大学医学院第二附属医院手术治疗的44例女性外阴病变患者的临床病理资料,包括鳞癌11例,鳞状上皮内瘤变(VIN)10例,尖锐湿疣9例及白色病变14例。采用免疫组化SP方法检测HPV16/18E6蛋白、HPV6L1蛋白的表达。结果①4组外阴病变患者年龄中位数依次为:外阴鳞癌61岁、VIN 45.5岁、尖锐湿疣24岁、外阴白色病44岁,尖锐湿疣组中位年龄低于外阴鳞癌组及白色病变组(P<0.05),其余各组间中位年龄无统计学差异。②HPV16/18E6蛋白在外阴白色病变组、尖锐湿疣组、VIN组及外阴鳞癌组的阳性率分别为2/14、3/9、3/10、8/11,外阴鳞癌组高于白色病变组(P<0.05),其余各组间无统计学差异;HPV6L1蛋白在外阴白色病变组、尖锐湿疣组、VIN组、外阴鳞癌组的阳性率分别为1/14、8/9、4/10、3/11,尖锐湿疣组高于其他各外阴病变组(P<0.05),其余各组间比较差异无统计学意义。③HPV16/18E6蛋白、HPV6L1蛋白在早期外阴鳞癌的阳性率均高于中晚期(P<0.05);HPV16/18E6蛋白、HPV6L1蛋白表达均与外阴鳞癌的组织学分级、年龄分组、绝经情况、产次不相关(P>0.05);HPV16/18E6蛋白与HPV6L1蛋白在外阴鳞癌中的表达不相关(P>0.05)。结论外阴病变的发病与年龄及HPV16/18、HPV6感染有关;外阴鳞癌中HPV16/18、HPV6的感染率与FIGO分期有关,HPV16/18感染率与HPV6的感染率无关。     

THE CONSTRUCTION AND EXPRESSION OF THE MURINE SCFV GENE IN E. COLI AGAINST HUMAN CERVICAL CANCER

Objective To obtain the gene of murine Single chain Fv fragment （ScFv） against haman cervical cancer and to express it in E. coli. Methods The variable region gene fragments of the heavy and light chains, which were amplified respectively using recombinant DNA techniques from CsA125 hybridama cells, were spliced together through a flexible linker to ScFv against human cervical cancer. The ScFv genes were then cloned into expression vector pCANTAB 5E and expressed in E. coli HB2151 and TG1 respectively. The soluble ScFv were characterized by SDSPAGE and Western blot. The antigen-binding activities of the soluble and phage displayed ScFv were assayed by ELISA and cell immunohistochemical analysis. Results The expressed ScFv antibodies were soluble and phage displayed. soluble ScFv secreted and expressed in E. coli HB2151 induced by IPTG were confirmed with SDS-PAGE, Western blot and ELISA. The specific binding capacity of the soluble and phage displayed ScFv to the surface associated antigen of human cervical cancer cell line was further confirmed with immunohistochemical studies. Conclusion The soluble and phage displayed ScFv expressed in E. coll against haman cervical cancer showed high, specific affinity for the cervical cancer cell line surface associated antigen.     

CONSTRUCTION OF HUMAN INTERLUEKIN-18 DNA VACCINE AND IT‘S EXPRESSION IN MAMMALIAN CELLS

Ｈｕｍａｎｉｎｔｅｒｌｅｕｋｉｎ 18(ｈＩＬ 18)ｉｓａｎｅｗｌｙｉｄｅｎ ｔｉｆｉｅｄｃｙｔｏｋｉｎｅｍａｉｎｌｙ ｐｒｏｄｕｃｅｄｂｙｍｏｎｏｃｙｔｅｓａｎｄｍａｃｒｏｐｈａｇｅｓ.ＴｈｅｃｙｔｏｋｉｎｅｃａｎｉｎｄｕｃｅＴｈ1ｃｅｌｌｓｔｏｓｅｃｒｅｔｅＩＦＮ γ ,ＩＬ 2ａｎｄＧＭ ＣＳＦ .Ｉｔｅｎｈａｎｃｅｓｔｈｅｃｙ ｔｏｔｏｘｉｔｙｏｆＮＫａｎｄＣＴＬｃｅｌｌ,ａｎｄａｌｓｏｒｅｄｕｃｅｓｔｈｅｌｅｖ ｅｌｏｆＩＬ 10 .Ｔｈｕｓ,ｔｈｅｒｅｉｓｐｏｔｅｎｔｉａｌｔｈｅｒａｐｅｕｔｉｃｖａｌｕｅｏｆ…     

子宫颈癌患者HPV16 E6,E7血清抗体检测

通过基因工程技术制备HPV16 E6,E7抗原,用免疫酶联吸附法检测了44例子宫颈癌病人和20例健康献血员血清HPV16 E6,E7抗体。结果表明,健康人血清HPV16 E6和HPV16 E7抗体阳性者分别占35％(7/20)和20％(4/20)。病人血清中,HPV16 E6抗体阳性率68.18％(30/44),但其反应滴度均值与健康人无显著差异。HPV16 E7抗体阳性率为58.18％(25/44),其阳性率及反应滴度均明显高于健康人。这提示,HPV16 E6抗体可能并非HPV16的型特异性抗体,而HPV16 E7抗体则有可能成为子宫颈癌发生的预报信号。     

THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

Objective To construct eukaryutic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humorul and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major rapsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, ETMxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry After BALB/c mice were vaccinated with various recombinant plusmids（pVAX1- L1-E6M3 or pVAX1-L1-E7M3 ） and immunie adjuvants （pLXHDmB7-2 or LTB） through different administration routes （intramuscular or intranasal） , the great cellular immune responses were produced us revealed by delayed-type hypersensitivity （DTH） and lymphocyte proliferation, and the expression of IL-4 and IFN- 7 cells in CD4^＋ and CD8^＋ subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8^＋ IFN-γ^＋ cells number, but CD4^＋ IL4^＋ cell number was higher in intranasul immunization groups. The immunization groups using pLXHDmB7.-2 as adjuvant were superior to other groups in immunorespouse. Conclusion These DNA vaccines produce remarkable cellular and humorul immune responses in the mouse and may provide us prophylatic and therapeutic candidates for HPV induced cancer treatment.     

THE CONSTRUCTION AND PRELIMINARY APPRAISEMENT OF HSV-2 gD GENE DNA VACCINE

Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｙｐｅ 2 (ＨＳＶ 2 ) ,ｏｎｅｋｉｎｄｏｆｄｏｕｂｌｅｓｔｒａｎｄｓＤＮＡｖｉｒｕｓ ,ｃａｎｃａｕｓｅｓｅｒｉｏｕｓｗｏｒｌｄｗｉｌｄｉｎｆｅｃｔｉｏｎｓ.Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｇｅｎｏｍ ｉｃＤＮＡｃｏｄｅｓｆｏｒａｐｐｒｏｘｉｍａｔｅｌｙ 80 ｐｒｏｔｅｉｎｓ .Ｍｏｓｔｏｒａｌｌａｒｅｅｘｐｒｅｓｓｅｄｄｕｒｉｎｇｌｙｔｉｃｉｎｆｅｃｔｉｏｎ ,ｍａｎｙａｒｅｆｏｕｎｄｉｎｉｎｔａｃｔｖｉｒｉｏｎｓａｎｄａｔｌｅａｓｔ 11ｈａｖｅｂｅｅｎｉｄｅｎｔ…     

THE DETECTION OF HPV16E6，E7 GENES IN TISSUES AND THEIR ANTIBODIES IN THE SERA OF PATIENTS WITH CERVICAL CARCINOMA

ＴＨＥＤＥＴＥＣＴＩＯＮＯＦＨＰＶ１６Ｅ６，Ｅ７ＧＥＮＥＳＩＮＴＩＳＳＵＥＳ　ＡＮＤＴＨＥＩＲ　ＡＮＴＩＢＯＤＩＥＳＩＮＴＨＥＳＥＲＡＯＦＰＡＴＩＥＮＴＳＷＩＴＨ　ＣＥＲＶＩＣＡＬＣＡＲＣＩＮＯＭＡＬｉｕＴｉａｎｊｕ；ＬｉｕＨｕａ；，ＳｕｎＹｉ；，Ｓ...     

人子宫颈癌中的HPV_(16)E_6mRNA、p~(53)、P~(21)~(ras)和c-myc蛋白表达

为进一步探讨子宫颈癌的分子发病机制，采用PCR、ISH和IHC方法对51例子宫颈癌组织的HPV16感染、HPV16E6mRNA、p53、p21ras和c－myc蛋白表达分布进行了研究。结果表明51例中有HPV16感染者38例，HPV16E6mRNA表达者35例，其阳性细胞主要分布在高分化癌巢之周边部或中、低分化癌之整个癌巢；p53、p21ras和c－myc蛋白阳性检出率分别为33．3％、60．8％和76．5％，其阳性细胞是弥漫或灶状分布。统计分析表明HPV16感染与P53、P21ras、c－myc蛋白积累间无关，但后三者间则显著相关。这提示癌基因活化或抗癌基因的失活及HPV16感染等因素之协同在子宫颈癌的发生发展中起重要作用。