禽传染性支气管炎病毒S1基因RT-PCR、RT-套式PCR扩增及其序列分析

【作者】 魏勇；

【导师】 王红宁； 文心田；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2001， 硕士

【摘要】 本研究采用RT-PCR（reverse translation-polymerase chain reaction）和RT-套式PCR（reverse translation-nest polymerase chain reaction）对7株禽传染性支气管炎病毒(IBV：M₄₁、Ma₅、SAIB₄、SAIB₁₄、SAIB_WJ、SAIB₆、SAIB₉)进行了S1基因的扩增，首次对四川地区分离的3株IBV的S1基因进行了序列分析。本研究利用SDS-蛋白酶K法提取7株IBV的基因组RNA。根据国际基因库Genbank注册的IBV呼吸型标准毒株(M₄₁株)的S糖蛋白基因全序列设计了内、外两对引物。用外引物对7株IBV的S1基因进行了RT-PCR扩增，其中M₄₁、Ma₅、SAIB₄、SAIB₁₄、SAIB_WJ5株获得了长约1.72kb特异条带，SAIB₆、SAIB₉ 2株未扩增出特异条带。7株IBV的RT-PCR扩增产物经稀释后用内引物进行RT-套式PCR扩增，7株都得到长约1.62kb的特异扩增条带，结果表明RT-套式PCR扩增较RT-PCR具有更高的敏感性。将SAIB₄、SAIB₁₄、SAIB_WJ三株IBVS1基因的RT-PCR特异扩增条带插入到pUC18质粒多克隆位点，转化DH_5α载体菌，筛选获得阳性克隆菌株。对克隆质粒中外源片段序列测定表明，所测序列内均含有长为1611bp的IBV S1结构基因，编码537个氨基酸，N-端有一编码18个氨基酸的信号肽序列。本研究首次将3株四川分离株SAIB₄、SAIB₁₄、SAIB_WJ的S1基因在国际基因库Genbank注册(注册号：SAIB₄为bankit404421、SAIB₁₄为bankit404417、SAIB_WJ为bankit404422)。与国际基因库Genbank注册的IBV M₄₁标准株S1基因序列比较，SAIB₁₄、SAIB₄、SAIB_WJ的S1基因与其核苷酸序列同源率分别为95.10％、79.24％、80.45％，相应的氨基酸序列同源率分别为91.44％、75.61％、76.16％。本研究丰富了IBV分子流行病学资料，为进一步研制IB DNA疫苗提供了基础材料。更多还原

【Abstract】 RT-PCR and RT-NPCR was employed to amplify 7 strain IBV (M41, Ma5, SAIB4, SAIB ~ SAIB w~, SAIB6, SAIB 9) , including two standard stains(M4 1, Ma5) and 5 strain isolates (SAIB4, SAIB 14, SAIB wj, SAIB6, SAIB9) . And for the first time, the sequence analysis of three IBV isolates from Sichuan Province in China had been done. Two pairs of primer were designed referring to the S glycoprotein (including Si and S2 glycoprotein) gene sequence of IBV M4, strain published in Genbank (Accession number: M21883). A specific fragment about 1.72kb was produced by RT-PCR from five stains (M41, Ma5, SAIB4, SAIB,4 and SAIBwj) respectively, while no band from the rest two strains (SAIB6 and SAIB9). Then a band about 1.62kb, was amplified by RT-NPCR with the inside primer from all of the 7 strains. It is shown that RT-NPCR is more sensitive than RT-PCR. The RI-PeR product of three isolates (SAIB4, SAIB ~ and SAIBw1) was cloned and sequenced. The result shows that, the coding region is 1611 bp in size, coding a 1 8-amino signal-peptide and a 519-amino mat-peptide. All of the three gene sequences have been accessed by Genbank (Accession number: SAIB4 is bankit 404421, 5MB ,~ bankit 404417, and SAIB wj bankit 404422). Comparison of the Si gene sequences of the three IBV isolates with IBV M~ strain, the nucleotide homology of SAIB ,4,SAIB4 and SAIBw~ is respectively 95.10%, 79.24% and 80.45%, and the correspondingly amino-acid homology equals to 91.44%, 75.6i%and 76.16%.更多还原