【作者】 田双喜；

【导师】 申之义； 关平原；

【作者基本信息】 内蒙古农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 本研究采用鸡胚绒毛尿囊膜接种培养的方法，对ILTV-NM98a株和ILTV-王岗株进行增殖。根据已发表的ILTV TK基因的核苷酸序列设计一对PCR引物，以增殖的两株ILTV的DNA为模板，分别对它们的TK基因进行PCR扩增。将回收的PCR产物连接到适当的质粒载体上，转化感受态大肠杆菌，通过筛选对ILTV TK基因的阳性克隆进行扩增培养。利用适当的限制性内切酶回收TK基因片段，并用地高辛进行标记，制备成核酸探针。 本研究中首次对ILTV-NM98a株的TK基因进行了克隆和序列分析，结果表明：ILTV-NM98a株TK基因的核苷酸序列与已发表的ILTV TK基因的核苷酸序列具有高度的同源性，两者之间仅相差4个核苷酸，同源性高达99.7％，从而证实了ILTV TK基因是高度保守的，为ILTV TK基因核酸探针的制备提供了有力的依据。 采用斑点杂交的方法，对ILTV TK基因核酸探针进行特异性和敏感性检测。结果表明：该种核酸探针具有高度的特异性，它仅与ILTV的DNA呈现阳性反应，而与新城疫病毒、传染性法氏囊病病毒和传染性支气管炎病毒的核酸等均呈阴性反应。该种探针具有高度的敏感性，能够检测到20pg的ILTV的DNA。更多还原

【Abstract】 In this study,ILTV-NM98a strain and ILTV-wanggang strain were multiplied in chorioallantois.A pair of primers were devised according to the nucleic acid sequence of ILTV TK gene and the DNA of multiplied virus was used as pattern to amplify the gene of Tk by polymerase chain reaction(PCR).The product of PCR was linked with suitable plasmid.Then,the recombined plasmid was converted to Escherichia coli.The converted Escherichia coli. Was multiplied and the TK gene was cloned.The cloned TK gene was retrieved by proper restrictive hemodynamics.The retrieved TK gene was labeled by digoxin according to the kit of labeling and detection of digoxin.Then,the specificity and sensitivity of TK gene probe were detected with dot blot hybridization.The sequence of TK gene of NM98a strain was analysed.The result of the analysis of TK gene’s sequence confirmed that autoploidy between TK gene of NM-98a strain and issued strain was 99.7%.The specificity of the nucleic acid probe was very strict.lt reacted positively with ILTV DNA only and it react negatively with the nuleic acid of NDV,BV and IBDV.The sensitivity of this kind of probe is very high.Ilt could even detect 20pg’s ILTV DNA.更多还原