摘要
本试验将乙型脑炎病毒（ＪＥＶ）的ＰｒＭ／Ｅ基因的ＨｉｎｄⅢ－ＢｇｌⅡ片段（２．１ｋｂ）插入到杆状病毒载体ｐＡｃＵＷ３１的ＢａｍＨＩ位点，使外源基因置于多角体蛋白启动子下游，构建成转移载体ｐＡｃＵＷ３１ＪＥ。以Ｌｉｐｏｆｅｃｔｉｎ作为共转染试剂，将纯化的ｐＡｃＵＷ３１ＪＥ与Ｂｓｕ３６Ⅰ线性化的杆状病毒ＡｃＭＮＰＶ·ＬａｃＺＤＮＡ共转染昆虫细胞ｓｆ９，经病毒蚀斑纯化技术和Ｘ－ｇａｌ与中性红双重染色技术，随机挑取了５个白斑并进一步纯化，用间接荧光抗体技术对挑取的５个白斑感染的ｓｆ９细胞染色，３个克隆在细胞核和细胞浆内呈现特异荧光，２个无特异性荧光。用地高辛标记ＰｒＭ／ＥｃＤＮＡ的ＨｉｎｄⅢ－ＢｇｌⅡ片段，制备了核酸探针，用该探针通过斑点杂交技术对５个白斑进行检测。结果发现，３个为重组的阳性克隆，２个为假阳性，该结果与荧光抗体染色的结果相一致。间接荧光抗体染色的结果也说明ｓｆ９细胞表达了所期望的产物，进而也说明了基因片段的正确性。ｐＡｃＵＷＪＥ转移载体的构建，为ｐＡｃＵＷＪＥ－ＰＰＶ的构建奠定了基础，为ＪＥＶ和ＰＰＶ二价重组苗的研究提供了保障。
A transfer vector,defined as pAcUW31JE,was constructed by inserting Hind Ⅲ Bgl Ⅱ fragment (2 1kb) of PrM/E cDNA of Japanese encephalitis virus (JEV)into BamHI site of baculovirus expression vector (pAcUW31) The PrM/E cDNA was placed under the control of polyhedrin promoter A co-transfection experiment was carried out with the purified pAcUW31JEDNA and wild-type baculovirus (AcMNPV LacZ) DNA which was linearized by Bsu36Ⅰ digestion,in which the Lipofectin was used as a co transfect reagent Plaquassays was conducted and 5 white plaques was picked for further purification and dualstaining with X gal and neutral red Three cloned sf9 cells showed the specific fluoresence by indirect immunofluoresence technique which confirmed that the protein of interest was present in the cytoplasm and nuclear of the sf9 cell infected with the recombinant baculovirus Two cloned sf9 cell did not show the specific fluoresence A PrM/E cDNA probe was prepared using the DIG Labeling and Detecting Kit Dot hybridization analysis of the 5 putative recombinant viral DNA using the probe indicated that 3/5 were positive,2/5 were false positive recombinants The result was the same as the result of the indirect immunofluoresence and the specific fluoresence also confirmed the reality of the gene fragment The construced transfer vector,pAcUW31JE,Lays the foundation for the construction of pAcUW31JE PPV,and the successful experiences of co transfection experiment by using the purified pAcUW31JE DNA and linearized AcMNPV LacZ DNA give an ensure for the study of dual valence vaccine of JEV and PPV
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
1998年第6期546-552,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金
关键词
乙型脑炎病毒
杆状病毒
PrM/E基因
猪病
Japanese encephalitis virus,Baculovirus expression system,Immunogen,Vaccine
