猪瘟病毒糖蛋白E0基因的克隆及其表达研究

MOLECULAR CLONING AND EXPRESSION OF E0 GENE OF THE CHINESE CLASSICAL SWINE FEVER VIRUS AND ITS LOCALIZATION IN HOST CELLS

用ＲＴ－ＰＣＲＡ法扩增分别获得了中国猪瘟病毒强毒石门株和免化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ，并克隆到ｐＧＥＭ Ｔ载体中测定其核着酸序列和推导出其对应氨基酸序列；结果表明这两个毒株间的Ｅ０基因核着酸序列同源性为９５．３％，氨基酸序列同源性为９４％，有１４个残基的差异；与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的免化弱毒Ｃ株相应序列进行比较，所测石门病毒核苷酸序列与上述各株对应序列的同源性分别为９８．０％、９７．１％、９２．７％、８６．８％和９５．４％；氨基酸序列同源性分别为９７．４％、９６．１％、９５．３％、９２．７％和９４．４％；所测兔化弱毒核着酸序列与上述各株对应序列的同源性分别为９５．２％、９４６％、９１．３％、８５．１％和９９．０％；氨基酸序列同源性分别为９３．１％、９２．３％、９１．４％、９０．５％和９８．７％；氨基酸序列分析结果还表明：上述各株病毒糖蛋白Ｅ０序列均含有与地衣类及植物核苷酸酶同样保守的两个氨基酸基序ＳＬＨＧＩＷＰＸ（Ｘ为Ｇ或Ｅ）和ＥＷＮＫＨＧＷＣ，基序中Ｈ为ＲＮａｓｅ催化活性关键氨基酸残基；将上述Ｅ０基因亚克隆至真核表达载体ｐＥＧＦＰ－Ｃ１。

EO gene fragments of chinese classical swine fever virus (CSFV) Shimen strain (a standard virulent strain) and hog cholera lapinized vaccine(HCLV) strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector, respectively. The nucleotide sequences of two fragments were sequenced by Sanger's method and the amino acid sequences were deduced. Sequence analysis shows the homologies of the nucleotide sequence and the deduced amino acid of EO gene between Shimen strain and HCLV were 95.3% and 94%, respectively. Compared with the corresponding regions of ALD. GEP. Brescia. Alfort strain and C strain sequenced by K J. M. Moormann, the nucleotide sequence homology of Shimen strain EO gene is 98.0%, 97.l%, 92.7%, 86.8% and 95.4%, and the amino acid sequence 97.4%, 96.l%, 95.3%, 92.7% and 94.4%, respectively; similarly, the nucleotide sequence homology of HCLV EO gene compared with above mentioned strains is 95.2%, 94.6%, 91.3%, 85.l% and 99%, and the amino acid sequence 93.l%, 92.3%, 91.4%, 90.5% and 98.7%, respectively. It has also been found that above mentioned strain's EO RNases, belonging to the RNases family consisting of several fungal and plant RNases, contain two conserved streches of 8 amino acids each, SLHGIWPX (X for G or E) and EWNKHGWC, which are spaced by 38 nonhomologous amino acids and which form the RNase active site, Histidine residues in both streches is essential for RNase catalysis. We subcloned 696bp of EO gene cDNA into baculovirus transfer vector and constructed successfully recombinant baculovirus expressing GST-EO by homologous recombination in Sf9 cells. Furthermore, we also constructed recombinant eukaryotic expression vector pEGry-EO containing EO gene in frame and transfected PK-15 cell by lipofectamine, the fluorescene microscopy detection indicated expressed EO protein mainly locate in plasma of PK-15 cells, which is the same as the results when PK-15 cells are infected by CSFV.