汉坦病毒Hunan03株S基因分子特征及核蛋白结构分析

The characteristics of hantanvirus Hunan03 strain S gene and the nucleoprotein structure prediction

目的测定汉坦病毒S基因编码区序列，分析和预测其编码的核蛋白结构，用于指导汉坦病毒诊断性抗原的筛选。方法对免疫荧光技术（IFA）阳性的鼠肺组织应用RT-PCR法扩增汉坦病毒S基因完整开放读码框（ORF），扩增产物克隆到pGM-T载体，转化E-coliTOPIO，通过蓝白斑试验和PCR筛选阳性克隆后进行基因序列测定；利用NCBI和Swiss—Prot／TrEMBL数据库中相关基因与蛋白结构分析软件包，如BLSAT、ProtParam、PredictProtein等对s基因的组成、结构、生物学特征及蛋白二级结构进行预测和分析。结果PCR扩增产物大小约1290bp；构建了pGM—T／S载体并测序成功；S基因序列中ORF全长1290个核苷酸残基，GC含量为44．11％，AT含量为55．89％，包含429个氨基酸（20种）；序列递交至GenBank后获得的登录号为JN712306；与原型株76．118（No．M14626）核苷酸同源性为83％，氨基酸同源性为98％，存在10个非特异性的变异位点；蛋白总平均疏水性为-0．405；二级结构中含3个跨膜区、4个非跨膜区，包含55％的螺旋结构，6．1％的折叠结构和38．9％的环结构。结论汉坦病毒S基因的测序和生物信息学的分析，揭示了核蛋白基因结构特征，为诊断性抗原的研究奠定了基础。

Objective To analyze the conding region of hantanvirus S gene and predict the struc- ture of nueleoprotein for diagnostic antigen study. Methods RT-PCR was used to amplify the S gene of hantanvirus Hunan03 strain after designing specific primers. The amplification product was cloned into pGM- T vector and then the recombinant vector was transformed into E. coli TOP10, gene sequencing was earried out after blue-white selection and PCR screening for positive clones. The database of NCBI and Swiss-Prot/ TrEMBL were used to predict and analyze the structure, biological characteristics and protein structures of S gene. Results The amplification product was about 1290 bp, the pGM-T/S vector was constructed and suc- cessfully sequenced, the whole length of the open reading frame （ORF） was composed of 1290 nueleotide residues, among them the GC content was 44.11% and the AT eontent was 55.89%, it was composed of 429 amino acids （20 kinds） ,the aceession number of the sequence submitted to GenBank was JN712306 ,its homology of nueleotides to the 76-118 strain was 83% and the homology of amino acids was 98% , ten non- specific variation sites were found. The grand average of hydropathicity was -0.405. There were three trans- membrane domains and four non transmembrane domains in the secondary structure of nucleoprotein inclu- ding 55% of helix structure, 6.1% of sheet structure and 38.9% of loop structure. Conclusion The bioinformatics analysis of Hunan03 strain S gene might be important for provide the substructure data to re- veal the significance of S gene characteristics on hemorrhagic fever renal syndrome （HFRS） prevention and control.