应用痘苗病毒载体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒（Ｒｈｅｓｕｓｒｏｔａｖｉｒｕｓ，ＲＲＶ）Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ＰＪＳＡ１１７５－ＶＰ４。应用磷酸钙沉淀技术将ＰＪＳＡ１１７５－ＶＰ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ存在下筛选蓝色蚀斑。经３代以上纯化和病毒增殖，获重组病毒Ｒ－ＶＪＳＡ１１７５－Ｖｐ４。蚀斑滴定其满度达到１５×１０１１ＰＦＵ／Ｌ。经核酸杂交试验证明所获得的重组痘苗病毒带有猴轮状病毒Ｖｐ４抗原基因。用重组病毒感染ＴＫ－１４３细胞（或Ｖｅｒｏ细胞），在感染后４８ｈ，用酶免疫法（ＥＩＡ）检测受染细胞上清液和细胞裂解液中表达的猴轮状病毒Ｖｐ４抗原基因均呈阳性反应。本试验为本研究室轮状病毒基因工程疫苗的一部分，为深入了解轮状病毒基因结构及其功能在方法学上奠定了必要的基础。     

人神经元特异性烯醇化酶蛋白表达及抗体制备

目的克隆和表达人的神经元特异性烯醇化酶(HuNSE)基因,制备HuNSE多克隆抗体,以期用于朊病毒病及相关疾病的临床诊断。方法经RT-PCR扩增HuNSE基因和测序验证后,将其克隆于原核表达载体pQE30,在大肠杆菌M15中诱导表达HuNSE蛋白,蛋白经Ni-NTA亲和纯化后,免疫兔子,所制备的抗血清用ELISA、Westernblotting和免疫组化鉴定。结果所表达的HuNSE蛋白相对分子质量约为22000,以其为抗原制备的HuNSE特异性抗血清具有良好的免疫反应性。Westernblotting和免疫组化结果显示抗血清可识别重组和不同哺乳动物脑组织中的NSE蛋白。结论NSE基因在大肠杆菌中获得了高效表达,所制备的NSE特异性抗血清可用于朊病毒病及其他神经退行性疾病的诊断和研究。     

利用昆虫-杆状病毒表达系统表达人乳头瘤病毒16型L1蛋白

目的 获得人乳头瘤病毒16型（Human papillomavirus type 16,HPV16)L1蛋白。方法 以pFB1为载体构建HPV16L1杆状病毒表达质 ,并感染昆虫细胞Sf9结果 收集27℃,培养72h的感染重组病毒的Sf9,提取细胞蛋白。经SDS－PAGE蛋白电泳分析,发现有一相对分子质量为56000的蛋白表达,Western blot证实所表达的蛋白为HPV16L1。结论 昆虫一杆状病毒表达系统可有效地表达HPV16L1蛋白。     

冷冻蚀刻技术的经验介绍

在超微结构的研究中除用超高压电镜进行活体观察外,近来又发展了冷冻蚀刻技术,可在冷冻状态下观察活体材料,且可研究细胞膜的分子结构。我们在日本电子公司(JEOL)JEE-4C真空喷镀仪的FED冷冻蚀刻装置上进行了该技术的实验,摸到点滴经验介绍如下。     

应用痘苗病毒体表达猴轮状病毒VP4抗原基因

把编码猴轮状病毒Ｖｐ４抗原的第４基因片段插入到痘苗病毒表达载体ｐＪＳＡ１１７５的Ｐ７．５启动子下游，构建成在痘苗病毒Ｐ７．５启动子调控下表达猴轮状病毒Ｖｐ４抗原基因的重组质粒ｐＪＳＡ１１７５－Ｖｐ４。应用磷酸钙沉淀技术将ｐＪＳＡ１１７５－Ｖｐ４ＤＮＡ转入ＴＫ－１４３细胞，在ＢＵＤＲ和Ｘ－ｇａｌ 存在下筛选蓝色蚀斑。     

FURTHER STUDIES ON THE ETIOLOGY OF AUTUMN INFANT GASTROENTERITIS

This article deals with further studies on the etiology of a larger series of autumn infant gastroenteritis (AIG) in Beijing in 1979. Fecal samples were collected from 126 and 52 cases in 2 hospitals. The positive rate of rotavirus detected by simple direct electron microscopy (EM) was 38.1To in l hospital and 19.2'70 in the other. Ultrasections were made from fecal sediment of the cases with negative findings by simple direct EM. The positive rate of rotavirus detected by ultrasection EM was 75.670 in 1 hospital and 5270 in the other. The total detec- tion rate obtained by the combined use of these 2 methods reached 84.970 in l hospital. In 1 hospital, adenovirus was found simultaneously with rotavirus in 3 and singly in 2. In the other hospital, adenovirus was found simultaneously with rotavirus in l. Paired sera were obtained from 49 cases, of which 37 ('75.5'/o) showed a 4-fold or higher rise in complement-fixing (CF) antibody to rotavirus, mostly a 32-fold or higher rise. Single convales- cent phase sera were obtained in 6 cases which all gave high rotavirus CF antibody titers. Fecal samples were inoculated into primary thesus monkey kidney monolayer cell cultures for virus isolation. Apart from isolation of a Type 19 ECHO virus strain from l case, addition of trypsin to the fecal samples, maintenance media or both showed no cytopathic changes. A crude extract of a rotavirus particle- positive fecal sample was fed t0 4 newborn pig- lets and one 3-month-old thesus monkey, none of which developed diarrhea. Fecal samples of 133 0f the 178 cases were cultured for bacteria. Only 8 cases (670) showed pathogenic Escherichia coli growth.