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.     

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。     

白血病瘤苗对小鼠细胞毒性T淋巴细胞作用的研究

目的　探讨自制的一种白血病疫苗对C57BL/ 6小鼠细胞毒性T淋巴细胞 (CTL)杀伤功能的影响。方法　建立白血病荷瘤小鼠模型 ,用自制的 3种不同的白血病疫苗进行预防或主动免疫治疗 ,用MTT比色法检测预防或治疗 2周、4周后小鼠CTL杀伤活性 ,并与对照组相比较。结果　①随着白血病细胞在小鼠体内的生长 ,小鼠的CTL杀伤功能受到严重抑制。②灭活肿瘤细胞 +IFA +细胞因子 (rGM CSF +rIL 2 +rIL 6)的肿瘤疫苗在提高小鼠CTL的杀伤功能方面 ,优于灭活肿瘤细胞 +IFA肿瘤疫苗 ,而仅含灭活肿瘤细胞的疫苗作用不明显。结论　灭活肿瘤细胞 +IFA +细胞因子 (rGM CSF +rIL 2 +rIL 6)的肿瘤疫苗可以激活以CTL为代表的特异性细胞免疫功能 ,此种疫苗在血液恶性肿瘤的特异性主动免疫治疗中很有潜力     

DIAGNOSTIC VALUE OF SERUM PROGRP31-98 IN PATIENTS WITH SMALL-CELL LUNG CANCER

Small celllungcancer (SCLC )isasubtyperepresentingapproximately 15 %to 2 0 %ofallpri marylungcarcinomaoccurringinChina .Ingener al,SCLCishighlysensitivetochemotherapyandhasafrequenttendencytometastasizeatanearlystage .Thus ,itisveryimportanttomakeanearlydiagnosisforthetreatmentofSCLC patients .NSEhaslongtermbeenusedasSCLCearlydiagnosismarkerinclinic[1] .However ,therelativelyhighfalse positiveratehasforcedthedoctorstolookforthemorespecificandsensitivemarker[2 ] .SincePro gastrin relea…     

HUMAN PAPILLOMAVIRUS TYPE 16 L1 PROTEIN CAN BE EXPRESSED IN LIVE ATTENUATED SHIGELLA FLEXNERI 5A STRAIN SH42

Cervicalcanceristhesecondmostcommon canceramongwomenworldwide,anditaccountfor thehighestmortalityindevelopingcountries.There areabout600000newcasesdiagnosedanditcauses aboutaquartermillionwomendeathperyear[1]. Highrisktypehumanpapillomavirusinfection (suc…     

IN VITRO CO-STIMULATORY ACTIVITY OF HUMAN B7.2(IgV+C)PROTEIN PRODUCED BY ENGINEERED BACTERIA

It was well known that "two signals" wereboth necessary to activate T lymphocytes. The firstsignal was provided by TCR--CD, which was en-gaged with antigen peptide--MHC complex, and thesecond, also named the costimulatory signal, wasprovided by the interaction between costimulatorymolecules B7. l and B7. 2 on antigen presenting celland its counter--receptor CD28/CTLA4 on T cell.The first signal alone without costimulation willlead T cell to anergy or apoptosis['~'2. B7. 2 mayplay a cri…     

突变型人肿瘤坏死因子-α工程菌发酵培养的实验研究

目的　优化基因重组人突变型 4 71肿瘤坏死因子 α(TNF α)工程菌的发酵和表达条件。方法　通过改变培养基的组分、pH值、培养温度和诱导表达的条件等 ,采用测定细菌生物量和蛋白表达量等方法 ,确定最适的发酵和表达条件 ,并考察工程菌中重组质粒的遗传稳定性。结果　突变型 4 71rhTNF α工程菌在 pH值 7.5的SOC培养基中 ,30℃培养、4 2℃诱导 5h时 ,其表达量最大。而且 ,所构建的重组质粒在工程菌DH5α中传代稳定 ,目的蛋白的表达不受影响。结论　优化了基因重组人突变型 4 71rhTNF α工程菌的发酵和表达的最适条件 ,为进一步开发人突变型 4 71rhTNF α奠定了基础     

沙眼衣原体D型MOMP基因的克隆和序列分析

目的　从D型沙眼衣原体培养物中克隆主要外膜蛋白(MOMP)基因。方法　利用细胞培养法扩增沙眼衣原体后,提取基因组为模板,经聚合酶链反应(PCR)扩增全长MOMP基因,并用酶切、PCR扩增及序列测定等方法对重组质粒进行鉴定。结果　成功扩增了MOMP基因,并插入克隆载体 pUCmT中。结论　MOMP基因的克隆为进一步开展沙眼衣原体的疫苗研究奠定了基础。     

THE ADHERENCE OF CARIOGENIC BACTERIA TO PELLICLE FORMED IN VITRO FROM MIXED AND DELIPIDATED SALIVA

Ｓａｌｉｖａｒｙｌｉｐｉｄｓ,ａｓａｓｏｒｔｏｆｏｒｇａｎｉｃｃｏｍｐｏｎｅｎｔｓｏｆｓａｌｉｖａ ,ｐｌａｙａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｏｒａｌｐｈｙｓｉｏｌｏｇｙａｎｄｐａｔｈｏｌｏｇｙ .Ｔｈｅｐｒｅｖｉｏｕｓｓｔｕｄｉｅｓａｂｏｕｔｓａｌｉｖａｒｙｌｉｐｉｄｓｆｏｃｕｓｅｄｏｎｉｔｓｏｒｉｇｉｎ ,ｉｔｓｒｅｌａｔｉｏｎｓｈｉｐｗｉｔｈｃａｒｉｅｓａｎｄｉｔｓｒｅｌａｔｉｏｎｗｉｔｈｓａｌｉｖａｒｙ ｇｌｙｃｏｐｒｏ ｔｅｉｎ[1-3 ] .Ｂｕｔｔｈｅｒｅａｒｅｆｅｗｓｔｕｄｉｅｓｏｎｓａｌｉｖ…     

增强型绿色荧光蛋白标记的Hela细胞株的建立

目的构建增强型绿色荧光蛋白(EGFP)标记的Hela细胞系。方法PCR和DNA测序鉴定pEGFP-C1真核表达质粒,采用Qiagen tip 500进行pEGFP-C1真核表达质粒DNA的提取和纯化;cellfectin转染Hela细胞,G418筛选,有限稀释法单克隆培养,荧光显微镜进行荧光检测EGFP的表达,采用激光共聚焦显微镜观测器检测荧光特性。结果PCR和DNA测序证实pEGFP质粒结构正确;在倒置荧光显微镜下,转染24h后,部分Hela细胞可见绿色荧光,转染72h时,大约80%Hela细胞内均可见绿色荧光。加入含G418的选择性培养基进行选择性培养,选择荧光较强的Hela细胞经有限稀释,持续筛选及克隆化培养,获得稳定表达绿色荧光的Hela细胞克隆,扩大培养并传至10代以上,将此细胞株命名为Hela-EGFP。激光共聚焦显微镜观察发现:在蓝光(-395nm)激发时,Hela-EGFP细胞绿色荧光激发波长为395nm,最大发射峰为509nm。结论Hela-EGFP细胞株具备稳定表达EGFP的能力,为实时可视化进行宫颈癌侵袭转移机制的研究奠定了基础。