摘要
血凝素 (HA)是禽流感病毒诱导机体产生保护性体液免疫反应的主要靶抗原。本研究的目的是克隆HA基因 ,为以后的表达及基因工程疫苗的研制奠定基础。收获接毒鸡胚的尿囊液 ,超速离心沉淀病毒 ,采用异硫氰酸胍法提取病毒RNA ,利用反转录_聚合酶链式反应 (RT_PCR)技术扩增出禽流感病毒 (H9N2亚型 )血凝素的cDNA。PCR产物加A尾后 ,用玻璃奶试剂盒回收纯化目的片段 ,通过T_A连接将加尾PCR产物连接到线状克隆载体pGEM_Teasyvector,转化DH5α感受态细胞 ,在含氨苄青霉素的LB平板上筛选阳性克隆 ,提取质粒 ,经酶切鉴定 ,对目的片段进行测序 ,结果获得了HA基因全长 ,约 1.7kb ,与已知序列进行同源性比较 ,同源性最高的可达 97% ,所克隆的基因为禽流感HA基因工程疫苗的研究奠定了基础。
Studies have proved that hemagglutinin (HA) is the main target Ag of chicken humoral immunity against AIV; It plays an important role in inducing key protective immunity. The purpose of this research is to amplify the HA gene. It will provide the basis for expression and engineering vaccine generation. We had obtained allantoic fluid from chicken embryo inoculated with AIV. Then we extracted the viral RNA using guandidinium isothiocyanate and amplified cDNA of HA by RT_PCR. We added the A_tail to the product of PCR, purified and extracted the target fragment by using glassmilk purification kit and cloned the product into pGEM_T easy vector by T_A ligation. Then the DH5 α competent cells were transformed with the recombinant plasimid. Consequently, the recombinant plasimid was amplified and extracted from the transformants and cut by Not I to test the inserting of DNA fragment. Sequencing the HA fragment of the positive clone showed that we have cloned the whole length of HA gene (about 1.7kb). Compared with the published HA sequences, the highest homology is 97%. The product provided a good foundation for the study of gene engineering vaccine of AI.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2001年第2期91-94,共4页
Chinese Journal of Preventive Veterinary Medicine
