汉滩病毒核蛋白的分段表达及抗原表位分析

在大肠杆菌中对汉滩病毒Ｓ基因４种不同长度片段的重组表达质粒进行诱导表达。结果表明表达的４种ＧＳＴ－ＮＰ融合蛋白均以不溶性包含体形式存在于茵体细胞内，表达量分别占菌体蛋白总量的２９－３６％，分子量分别约为７２ｋＤ、６６ｋＤ、５４ｋＤ和４４ｋＤＤ。Ｗｅｓｔｅｒｎ ｂｌｏｔ显示５４ｋＤ和７２ｋＤ融合蛋白用酶标记汉滩病毒ＮＰＭｃＡｂｌＡ８和抗ＧＳＴ ＭｃＡｂ ３Ｃ１１染色呈阳反应。６６ｋＤ和４４ｋＤ融合蛋

Expression of Truncated HTNV Nucleoprotein and Analysis of Antigenic epitope

The expressing vector carring various truncated fragments of S gene of HTNV strain 76 118 was constructed and the vector efficienly expressed in E.coli . The result demonstrated that the GST NP fusion proteins exist in the form of inclusion bodies in E.coli , the expressing amount accounted for 29 36% of the total proteins, and the molecular weights were 72 kD, 66 kD, 54 kD and 44 kD, respectively. Western blot showed that all of the four fusion proteins were positive stained with HPR labeled anti GST McAb 3C11, but only the 72 kD and 54 kD fusion proteins were positive stained with HRP labeled anti NP McAb 1A8. Four recombinant NP (rNP), which molecular weights were 44 kD, 40 kD, 26 kD and 16 kD respectively, were obtained by removing GST from purified GST NP fusion proteins with thrombin. Mapping of antigenic epitope was done by 19 strains McAb. The result showed that 72 kD fusion protein could react with 13 strains McAb, which was same as authentic NP of HTNV. The truncated fusion protein (deleting aa 1 37 at N teminal and aa 402 429 at c terminal) expressed by S1.1 kb fragment and S 0.5 kb fragment (deleting aa 1 274 at N terminal) did not react with all 19 strains McAb. The truncated fusion protein (deleting aa 275 429 at c terminal) expressed by S 0.7 kb fragment reacted with 5 strains of group specific McAb, which suggested that antigenic epitopes on NP of HTNV were located in the N terminal of NP and distribution of group specific and type specific antigenic epitope showed some regional.