本研究参照已发表的IBV基因序列设计合成了一对引物,经反转录-聚合酶链式反应(Reverse Transcription-polymerase Chin Reaction,RT-PCR)技术扩增得到我国地方分离株LX4 mRNA_5和mRNA_6的全部cDNA大小约1.7kb的片段。将该片段克隆并进行序列测定,与国内外部分参考毒株的相应序列比较分析发现:LX4 5a基因与已发表的国内外IBV毒株之间同源性很低,而这些参考毒株间5a基因同源性却很高(推导的氨基酸同源性在90%以上)。5b基因与国内分离株D971同源性最高。在所选的12株参考毒株中,LX4 N基因推导氨基酸同源性与国内分离株SD/97/02最高(94%),与HB次之(93%),与其他毒株核苷酸和推导氨基酸同源性在89%和92%以下。对已报道的N基因所编码蛋白的三个保守碱性区同源性比较发现,LX4在第66-88位与Beau、M41、H52及FIB同源性均为100%。在第181-234位,推导氨基酸同源性与SD/97/02最高(94%)。在第334-373位推导氨基酸同源性与H120和ZJ971推导氨基酸同源性均为93%,与其他毒株在82%以下。同时还发现在c末端350-409位,LX4与H120推导氨基酸的同源性为100%,与HB次之(98%),与其他毒株在95%以下。以上研究结果提示:我国地方分离毒株LX4 mRNA_5和mRNA_6 cDNA序列与国内其他分离株最高,表明与国内其他分离株具有较近的遗传衍化关系。但在N基因序列比较及对N基因编码蛋白的局部功能区比较分析发现,LX4与H52和/或H120存在很高的同源性,这表明LX4的存在可能与我国应用IBV疫苗株H52和H120有一定的联系。

According to the published IBV gene sequence, a pair of specific primers were designed and synthesized. A fragment of 1.7kb including mRNAs and mRNA_6 cDNA of infectious bronchitis virus LX4 was amplified, cloned and sequenced. Then the nucleotide and the deduced amino acid sequence of this fragment were analyzed. The results showed that N gene of IBV LX4 sharedthe highest identity with SD/97/02 and HB that were both isolated in China. However, the identity is different in three conservative basic regions of N protein. The deduced amino acid sequence of LX4 shared the highest identity with M41,Beau, H52 and HB in position 66-88, with SD/97/02 strain in position 181-234 and with H120 and ZJ971 in position 334-373 respectively. Meanwhile, we found that LX4 and H120 showed completely identical in position 350-409 both in corresponding nucleotide and deduced amino acid. All these data above showed that mRNA_5 and mRNA_6 cDNA of IBV LX4 shared more characteristics with other isolates in China than the aboard. In a