猪传染性胃肠炎病毒SC-1株的分离与基因7的克隆分析

从四川某猪场采集疑似病例的腹泻粪样中分离到1株病毒,该病毒经过ST细胞传代培养盲传至第4代时出现病变。通过荧光抗体染色、病毒中和试验、TCID50试验、核酸类型鉴定,结合样品正染后在电镜下的观察结果,确定该毒株为猪传染性胃肠炎病毒,命名为TGEV SC-1株。参照GenBank中收录的TGEV基因7的序列设计了1对特异性引物,以SC-1株的细胞增殖毒的总RNA为模板,通过RT-PCR扩增获得了大小约为303 bp的基因片段,包括完整的基因7序列片段（237 bp）,在GenBank上的登录号为DQ437507。核苷酸和推导的氨基酸序列比较分析结果表明,该毒株与其他毒株的同源性均在92.4%以上,TGEV SC-1株与美国的PURDUE和中国的TH-98毒株亲缘关系最近;与日本各分离毒株、中国TS株、美国Miller株、中国台湾TFI株和英国96-1933株的同源性较低。TGEV基因7在密码子使用的选择上,存在较大的偏向性;基因7编码蛋白在两端存在有明显的疏水性区域,在2～24和56～75位氨基酸残基处存在跨膜结构。

Isolation of SC-1 strain of transmissible gastroenteritis virus of swine and characteristic analysis of gene 7

One transmissible gastroenteritis virus(TGEV) strain designated SC-1 was isolated from feces samples collected from piglets manifesting serious diarrhea after 4 blind passages on ST monolayer cells.The piglets were from certain pig farm in Sichuan province.Cytopathogenic effect(CPE) occurred obviously on the fourth and thereafter ST monolayers.FA identification,TCID50,liposolubility reagent sensitivity experiment,type of nucleic acid experiment were also tested.The morphology of the virions in ST monolayers was also observed under the electron microscope.Then the fragment including gene 7 was amplified with method of RT-PCR based on a pair of special primers according to sequence of gene 7 from GenBank and the total RNA extracted from the supernatants from cell culture.The fragment of about 300 bp,was cloned into plasmid pMD18-T vector and identified by means of PCR,restriction enzymes digestion and sequencing.The gene 7 of 237 bp was submitted to GenBank with the accession number of DQ437507.Bioinformatics analysis based on nucleic sequence and inferred amino acids showed that identity of gene 7 of SC-1 isolate was above 92.4% as compared with American PURDUE strain and Chinese TH-98 strain,and was below 92.4% as compared with Japanese strains,Chinese TS strain,American Miller strain,Chinese Taiwan TFI strain and England 96-1933 strain.Gene 7 owned bias in usage of the codon.Hydrophobicity areas distributed in two terminates of the peptide of protein 7 had been predicted,transmembrane structure also had been predicted in the presence of position 2 to 24 and 56 to 75 amino-acid residues.