禽传染性支气管炎病毒弱毒疫苗株H52反向遗传株的构建

Construction of reverse genetic strain of IBV aettnuated vaccine strain H52

摘要:

IBV H52基因组全长27.6kb，分13个1.3-2.9kb的片段扩增H52全长cDNA,额外扩增N-3’片段，并克隆到载体PMD19-T上.利用No see’m技术在每个片段中引入BsaI或BsmBI酶切位点，D1片段中引入无义突变位点，在5‘UTR端和N-3’引入T7启动子，3’UTR端引入Poly（A）尾.将克隆好的13个片段酶切，纯化，体外无缝连接为全长cDNA.将全长cDNA及N-3’体外转录获得它们的转录体，将转录体共转染BHK-21细胞，48h后收获细胞液，将细胞液接种10日龄SPF鸡胚获得反向遗传株H52-R.在鸡胚中盲传5代后，通过RT-PCR对无义突变位点进行扩增及序列分析，在反向株H52-R中发现引入的无义突变位点.对反向株H52-R的一些生物学特性如HA、EID50等测定发现，H52-R的这些生物学特性与亲本株H52相似.

Abstract:

IBV H52 has a genome of approximately 27.6 kb in length, thirteen 1.3-2.9 kb fragments contiguously spanning the virus genome and the N-3’ fragment were amplified and cloned into PMD19-T. Bsa I or BsmB I restriction enzyme sites were introduced into each fragment base on “No see’m” ligation strategy, a silentmutation site was introduced into the D1 fragment and the T7 promoter was incorporated to the 5’UTR and N’3 fragments, the Poly(A) tail was incorporated to the 3’UTR. The complete length cDNA was acquired by seamless connection in vitro. Consequently, the full-length genomic cDNA and N’3 fragment was transcribed and transfected into BHK-21 cells by electroporation to rescue the virus. 48 h post transfection, the cultures of transfected BHK-21 cells was harvested and inoculated into 10-days old SPF embryonated eggs (ECE) to replicate the rescued virus. After five passages, the silentmutation site which was introduced artificially in the tenth (D1) fragment of the rescued virus (H52-R) was detectable, revealed that the H52-R virus was successfully rescued. We characterized the EID50 and HA titers of H52-R and confirmed that they were similar to its parent strain H52.