中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
8期
725-729
,共5页
蔡亮%高立冬%刘运芝%覃迪%刘建高%胡世雄%刘富强%湛志飞%曾舸%刘佳惠%张红
蔡亮%高立鼕%劉運芝%覃迪%劉建高%鬍世雄%劉富彊%湛誌飛%曾舸%劉佳惠%張紅
채량%고입동%류운지%담적%류건고%호세웅%류부강%담지비%증가%류가혜%장홍
汉坦病毒%S基因%分子特征%二级结构%预测
漢坦病毒%S基因%分子特徵%二級結構%預測
한탄병독%S기인%분자특정%이급결구%예측
Hantanvirus%S gene%Molecular characteristics%Secondary 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.coli TOP10,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1290 bp;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.
目的 測定漢坦病毒S基因編碼區序列,分析和預測其編碼的覈蛋白結構,用于指導漢坦病毒診斷性抗原的篩選.方法 對免疫熒光技術(IFA)暘性的鼠肺組織應用RT-PCR法擴增漢坦病毒S基因完整開放讀碼框(ORF),擴增產物剋隆到pGM-T載體,轉化E.coli TOP10,通過藍白斑試驗和PCR篩選暘性剋隆後進行基因序列測定;利用NCBI和Swiss-Prot/TrEMBL數據庫中相關基因與蛋白結構分析軟件包,如BLSAT、ProtParam、PredictProtein等對S基因的組成、結構、生物學特徵及蛋白二級結構進行預測和分析.結果 PCR擴增產物大小約1290 bp;構建瞭pGM-T/S載體併測序成功;S基因序列中ORF全長1290箇覈苷痠殘基,GC含量為44.11%,AT含量為55.89%,包含429箇氨基痠(20種);序列遞交至GenBank後穫得的登錄號為JN712306;與原型株76-118(No.M14626)覈苷痠同源性為83%,氨基痠同源性為98%,存在10箇非特異性的變異位點;蛋白總平均疏水性為-0.405;二級結構中含3箇跨膜區、4箇非跨膜區,包含55%的螺鏇結構,6.1%的摺疊結構和38.9%的環結構.結論 漢坦病毒S基因的測序和生物信息學的分析,揭示瞭覈蛋白基因結構特徵,為診斷性抗原的研究奠定瞭基礎.
목적 측정한탄병독S기인편마구서렬,분석화예측기편마적핵단백결구,용우지도한탄병독진단성항원적사선.방법 대면역형광기술(IFA)양성적서폐조직응용RT-PCR법확증한탄병독S기인완정개방독마광(ORF),확증산물극륭도pGM-T재체,전화E.coli TOP10,통과람백반시험화PCR사선양성극륭후진행기인서렬측정;이용NCBI화Swiss-Prot/TrEMBL수거고중상관기인여단백결구분석연건포,여BLSAT、ProtParam、PredictProtein등대S기인적조성、결구、생물학특정급단백이급결구진행예측화분석.결과 PCR확증산물대소약1290 bp;구건료pGM-T/S재체병측서성공;S기인서렬중ORF전장1290개핵감산잔기,GC함량위44.11%,AT함량위55.89%,포함429개안기산(20충);서렬체교지GenBank후획득적등록호위JN712306;여원형주76-118(No.M14626)핵감산동원성위83%,안기산동원성위98%,존재10개비특이성적변이위점;단백총평균소수성위-0.405;이급결구중함3개과막구、4개비과막구,포함55%적라선결구,6.1%적절첩결구화38.9%적배결구.결론 한탄병독S기인적측서화생물신식학적분석,게시료핵단백기인결구특정,위진단성항원적연구전정료기출.
Objective To analyze the conding region of hantanvirus S gene and predict the structure of nucleoprotein 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 carried 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 successfully sequenced,the whole length of the open reading frame (ORF) was composed of 1290 nucleotide residues,among them the GC content was 44.11% and the AT content was 55.89%,it was composed of 429 amino acids (20 kinds),the accession number of the sequence submitted to GenBank was JN712306,its homology of nucleotides to the 76-118 strain was 83% and the homology of amino acids was 98%,ten nonspecific variation sites were found.The grand average of hydropathicity was-0.405.There were three transmembrane domains and four non transmembrane domains in the secondary structure of nucleoprotein including 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 reveal the significance of S gene characteristics on hemorrhagic fever renal syndrome (HFRS) prevention and control.