绵羊肺腺瘤病毒NM株前病毒基因组的克隆与全序列分析

Cloning and Sequencing Analysis of the Complete Genome of Jaagsiekte Sheep Retrovirus Inner Mongolia Strain

本研究参照GenBank中已发表的绵羊肺腺瘤病毒的基因组全序列,设计合成8对引物,从内蒙古某羊场自然感染绵羊肺腺瘤病的病肺肿瘤组织中提取总DNA为模板,对JSRV-NM株基因组分8段进行PCR扩增,产物分别为8个(531bp,888bp,949bp,944bp,1428bp,947bp,1836bp,538bp)基因片段,将其分别克隆入pMD-18T载体中进行双向测序并拼接序列,获得完整的JSRV-NM株前病毒基因组全序列。结果表明,JSRV-NM株前病毒基因组全长7430bp,有相互重叠的4个较长的开放阅读框(ORF),分别代表gag、pro、pol和env基因。与绵羊肺腺瘤病毒Ⅰ型即南非代表株(NC-001494)和绵羊肺腺瘤病毒Ⅱ型即美国代表株(AF105220)的核苷酸同源性比较分别为90.4%和90%,推导出的氨基酸同源性分别为90%和89.1%。分析JSRV-NM株基因组结构,发现在LTR的上游和下游都具有外源性exJSRV特有的ScaⅠ酶切位点,在gag基因编码的NC区发现有2个较典型的“胱氨酸—组氨酸序列”,可形成锌指结构。在env基因编码的TM区有特异性的“YXXM”基序。用地高辛标记外源性exJSRV特异的JSRV-2片段制成探针,原位杂交法检测自然感染绵羊肺腺瘤病(OPA)的病肺组织中JSRV-NM的RNA及前病毒DNA,结果表明OPA患羊肺肿瘤细胞的胞浆和核内都有JSRV-2基因mRNA的表达,说明JSRV-NM株是具有致瘤作用的外源性反转录病毒。这是我国首次报道的绵羊肺腺瘤病毒的基因组全序列。

In order to amplify the complete genome of Jaagsiekte sheep retrovirus （JSRV） Inner Mongolia Strain, eight pairs of primers were designed based on Genbank sequences. Eight fragments were obtained by PCR and were cloned into the pMD-18 T vectors. The recombinant plasmids were sequenced and the complete sequences were analyed with DNA Star. The results showed that the genome was 7430bp in length and contained four overlapping open reading frames of the gag, pro, pol and env genes. The nucleotide and amino acid sequences of NM strain were compared with the sequences of South Africa strain （type I, Accession No. NC-001494） and USA strain （type Ⅱ, Accession No. AF105220）. The nucleotide and amino acid identities were 90.4% and 90%, 90% and 89.1%, respectively. Two zinc fingers were found in the region of NC in the predicted amino acid sequence. A Sca Ⅰ restriction site in gag was found in the sheep genome, The ＂YXXM＂ motif was found in the region of TM, which are reliable molecular markers for the infectious exogenous virus. The exJSRV-2 specific DNA fragment probe was labeled with digoxigenin （Dig） and the Jaagsiekte retrovirus NM strain RNA and proviral DNA in the lung tissue of OPA were detected by in situ hybridization. The results showed JSRV-NM mRNA and provirus DNA in sheep lung tumor cells had very strong hybridization signals while the control group did not have a positive signal. This is the first nucleotide sequence of exJSRV reported in China.