猴副流感病毒检测方法的建立与初步应用

【作者】 吴凡；

【导师】 贺争鸣；

【作者基本信息】 中国药品生物制品检定所 ， 免疫学， 2006， 硕士

【摘要】 猴副流感病毒SV5(Simian parainfluenza virus 5)是猴群中普遍存在的一种RNA病毒，也是猴源细胞培养物中常见的污染物。为保证实验动物质量和利用猴源性细胞制备的人用生物制品的质量和安全性，有必要在实验动物和生物制品的质量控制中对该病毒进行检测。本文旨在建立针对猴副流感病毒(Simian parainfluenza virus 5，SV5)的分子生物学和血清学检测方法，并对相关生物材料和制品进行检测。 为建立猴副流感病毒SV5的PCR检测方法，针对SV5基因组最保守的SH基因片段(292bp)按照引物设计原则设计一对引物，从SV5感染Vero细胞培养液中提取总RNA，用随机引物反转录合成cDNA，以设计的特异性引物进行PCR，能够扩增出一条约237bp的特异性目的条带，将纯化的PCR产物连接到pGEM-T载体，测序得到克隆基因的核酸序列，将该序列经BLAST程序搜索，结果证实为SV5基因组SH基因的特异片段。通过对反转录体系中的dNTP浓度，AMV逆转录酶浓度以及PCR反应体系中的dNTP，镁离子，循环次数，退火温度，引物浓度、Taq酶浓度进行梯度实验，确定了RT-PCR检测方法的最佳工作条件。对已知浓度为0.21μg／μl的RNA样本做倍比稀释进行RT-PCR扩增，以鉴定RT-PCR方法的敏感性，结果显示能够检测到最小RNA浓度为2.1ng／μl。用所建立的RT-PCR方法对来源于北京生物制品研究所的实验猴(购于四川成都)组织和国内六个生物制品研究所生产的普通级地鼠以及北京实验动物研究中心生产的SPF级地鼠组织、猴源性生物制品(脊髓灰质炎减毒活疫苗)以及五种常用的猴肾传代细胞进行检测，结果在上述样品中均未检测到SV5核酸序列。 为检测实验用猴群中抗SV5抗体分布情况，利用中国医学科学院实验动物研究所提供的猴抗SV5阳性血清建立了针对猴血清的IFA和IEA两种SV5抗体检测方法。为检测地鼠群中SV5抗体分布群况，我们利用细胞培养的方法制备了SV5抗原并免疫SPF地鼠获取了地鼠抗SV5阳性血清，建立了针对地鼠血清抗体的IEA和IFA两种检测方法。通过浓度梯度滴定，分别确定了一抗和二抗的最佳工作浓度。将阳性血清进行浓度梯度稀释并进行检测，确定了两种方法的敏感度。用所建立的IFA和IEA两种方法分别检测来源于两个来源(北京协尔鑫生物资源研究所和海南金港生物技术有限公司)五个批次的614份猴血清样品，结果在其中一个批次的335份样本中检出89份阳性血清。用所建立的IFA更多还原

【Abstract】 Simian parainfluenza virus 5 (SV5) is a type of RNA virus which is an ubiquitous virus in simian’s, and also is a frequent contamination from simian’s cell cultures. To guarantee the quality of laboratory animal productions, and the quality and safety of biological products from simians for human use, it is necessary to detect SV5 in the process of quality control of laboratory animals and biological products. The purpose of our research work is to establish the methods based on molecular biological and serological technologies to detect SV5 in related biological products.To establish the PCR method for the detection of SV5, according the primer designing principle, a pair of primers were designed to aim directly at the SH gene (292bp), which is the most conservative gene fragment in the SV5 genome. Viral RNA was extracted from the Vero cell culture infected with SV5, and cDNA was reverse-transcripted by using Random Primer as 3’ out primer. An approximately 237bp idio-fragment was amplified from the cDNA by the designed primers. The amplified fragment was sequenced and proved to be the SH gene sequence of SV5 genome. The conditions of the RT-PCR were then optimized by a series of gradient experiments. Sensitivity of the method was determined as the minimum RNA concentration of 2.1ng/μl. For initial application of the method, samples including simian organ from Beijing Research Institute of Biological Products, ordinary hamster’s organ from six domestic research institute of biological products, SPF hamster’s organ from Beijing Lab Animal Center, biological products from simian (OPV), and five commonly used passage cells from simian kidney, were tested by the new method for the SV5 nucleotide gene.Results came out that no SV5 nucleotide gene were detected in above specimens.To detect the distribution conditions of SV5 antibody in laboratorial simians, two antibody detection methods, IFA and IEA, were established using the simian SV5-positive serum provided by Lab Animal Research Institute of China Academy of Medical Science. SV5 antigen was prepared by cell culture method, and the hamster’s SV5 positive serum was prepared by immunizing hamsters. Then the sensitivity of the two methods was determined. The two new methods were initially applied on simian serum samples and hamster serum samples from different sources. Result came out that 89 of 335 sepcimens positively reacted in total 614 simian serum soecimens,and no positive reaction in total 119 hamasters’ serum specimens.更多还原