Dybing J K, Jackwood D J
Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691, USA.
Avian Dis. 1996 Oct-Dec;40(4):931-7.
The variant MD strain of infectious bursal disease virus (IBDV) was examined using restriction fragment length polymorphism (RFLP) and the molecular variation was compared with published sequences of both variant and classic IBDV strains. Viral RNA was transcribed into cDNA using reverse transcriptase and then amplified by the polymerase chain reaction (PCR). Three separate but overlapping fragments of 1603 bp, 1496 bp, and 1066 bp containing the entire coding region for VP2, VP4, and VP3, respectively, were amplified. These amplified products were initially cloned using the TA Cloning kit and further subcloned into the pGEM-3Zf and pUC19. Eight restriction enzymes, PstI, BamHI, AvaI, EcoRI, HindII, XmaI, SmaI, and XbaI, were tested for their ability to digest the MD PCR products. The resulting RFLP was analyzed and compared with the published genome segment A sequences of the classic IBDV strain STC and the variant IBDV strain GLS. Differences in restriction fragment profiles were observed after digestion with BamHI, EcoRI, XmaI, and SmaI, with the absence of the EcoRI site in both variant strains, GLS and MD. The MD PCR products lacked both XmaI and SmaI sites, which were present in both STC and GLS. The MD PCR products contained a BamHI site within the hypervariable region of VP2, which is present in STC but not in the variant GLS. An additional BamHI site was identified in the VP4 gene of MD and GLS but not in STC.
使用限制性片段长度多态性（RFLP）技术对传染性法氏囊病病毒（IBDV）的变异MD株进行检测，并将其分子变异与已发表的变异株和经典IBDV株序列进行比较。使用逆转录酶将病毒RNA转录为cDNA，然后通过聚合酶链反应（PCR）进行扩增。分别扩增出三个独立但重叠的片段，长度分别为1603 bp、1496 bp和1066 bp，分别包含VP2、VP4和VP3的整个编码区。这些扩增产物最初使用TA克隆试剂盒进行克隆，然后进一步亚克隆到pGEM-3Zf和pUC19中。测试了八种限制性内切酶，即PstI、BamHI、AvaI、EcoRI、HindII、XmaI、SmaI和XbaI，它们消化MD PCR产物的能力。对所得的RFLP进行分析，并与经典IBDV株STC和变异IBDV株GLS已发表的基因组A片段序列进行比较。用BamHI、EcoRI、XmaI和SmaI消化后观察到限制性片段图谱存在差异，变异株GLS和MD中均不存在EcoRI位点。MD PCR产物既缺乏XmaI位点也缺乏SmaI位点，而这两个位点在STC和GLS中均存在。MD PCR产物在VP2的高变区内含有一个BamHI位点，该位点存在于STC中但不存在于变异株GLS中。在MD和GLS的VP4基因中鉴定出一个额外的BamHI位点，但在STC中未发现。
