表达H5亚型禽流感病毒HA蛋白的重组禽痘病毒的构建

Construction of recombinant folwpox virus expressing HA protein of H5 subtype avian influenza virus

为构建表达H5亚型禽流感病毒(AIV)A/Duck/Anhui/1/06(H5N1)(简称AH/06)HA蛋白的重组禽痘病毒,本研究利用感染-转染的方法通过转移载体pSY681-gfp-gpt将AIV株AH/06的HA基因同源重组到禽痘病毒(FPV)中,并利用gfp和gpt双重筛选标记在鸡胚成纤维细胞中经过多轮筛选,得到纯化的重组禽痘病毒(rFPV-gfp-gpt-AHHA)。通过PCR、序列测定和western blot的方法对rFPV-gfp-gpt-AHHA进行鉴定和抗原活性分析。结果表明AH/06的HA基因稳定的重组到rFPV-gfp-gpt-AHHA中,其表达的HA蛋白能够与AH/06的阳性血清反应,显示出了良好的抗原性。同时病毒的生长曲线也显示外源HA基因的插入并没有影响亲本病毒的复制。这些结果为进一步的免疫效力研究奠定了基础。

To construct recombinant fowlpox virus （FPV） expressing HA protein of H5 subtype avian influenza virus （AIV）, the HA gene from A1V A/Duck/Anhui/1/06（H5N1） was amplified by PCR and inserted into the transfer vector pSY681-gfp-gpt of FPV. The recombinant FPV （rFPV-gfp-gpt-AHHA） was generated by transfection of the recombinant transfer vector into the quail origin FPV pre-infected chicken embryo fibroblast and purified for several cycles of selection by using Marker genes of gfp and gpt. The rFPV-gfp-gpt-AHHA was identified by PCR, sequencing and western blot. The results proved that HA gene of AIV was integrated and expressed in the recombinant FPV. One-step growth curve showed that replication of rFPV-gfp-gpt-AHHA was similar to the parent FPV. However, the protection of rFPV-gfp-gpt-AHHA needs to further evaluate in chickens against AIV infection.