鸡肾型IBV分离株M基因的克隆与特性分析

Cloning and characterization of matrix glycoprotein gene of nephrotropic avian infectious bronchitis virus

依据NCBI所登录的鸡传染性支气管炎病毒的M基因序列设计了一对引物,应用TRIzol试剂盒对8个肾型鸡传染性支气管炎病毒陕西地方分离株进行总RNA的提取,以所得到的RNA为模板,用RT-PCR对M基因进行扩增;其目的条带回收提纯后,与PMD18-T克隆载体进行连接,转化到DH5α宿主菌,并经药物抗性筛选、PCR及酶切鉴定,将所筛选鉴定出的阳性质粒进行测序,最后对测序结果进行分析。结果表明:陕西地方8个毒株(BJ2、FF2、YL2、B01、G5、YX、WH、WG)M基因长约为650 bp,其中YX、WH株本身无BamH 1酶切位点。WH与其它分离株的核苷酸同源性为91.8%～92.6%,而其它的分离株间的同源性为98.8%～99.8%。8个毒株与参考株的核苷酸同源性为74.9%～99.8%。其N端90 aa肽段与对照株相比,不同之处表现为高亲水性氨基酸取代低亲水性氨基酸,这会使其具有更好的抗原性。

The M genes of 8 infectious bronchitis virus （IBV） isolates from shan＇xi were amplified by RT-PCR using a pair of primers designed according to the published Matrix glycoprotein gene sequence of IBV. The PCR products were cloned into PMD18-T vector and sequenced. Analysis of the M gene sequences showed that these local isolates shared 98.8 %-99.8 % nucleotide homology, The WH isolate was slightly divergent and shared 91.8 %-92.6 % identity with the others. Comparing to reference strains, the local isolates shared 74.9 %-99.8 % sequence identity with the published sequences. Furthermore, the amino acids in the N-terminal 90 aa region of the local isolates were substituted by the amino acids with high hydrophilicity, indicating a better antigenity of M gene in these isolates.