CAJ | 학술논문

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-T easy vector，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。
혈응소（HA）시금류감병독유도궤체산생보호성체액면역반응적주요파항원。본연구적목적시극륭HA기인，위이후적표체급기인공정역묘적연제전정기출。수획접독계배적뇨낭액，초속리심침정병독，채용이류청산고법제취병독RNA，이용반전록-취합매련식반응（RT-PCR）기술확증출금류감병독（H9N2아형）혈응소적cDNA。PCR산물가A미후，용파리내시제합회수순화목적편단，통과T-A련접장가미PCR산물련접도선상극륭재체pGEM-T easy vector，전화DH5α감수태세포，재함안변청매소적LB평판상사선양성극륭，제취질립，경매절감정，대목적편단진행측서，결과획득료HA기인전장，약1.7kb，여이지서렬진행동원성비교，동원성최고적가체97%，소극륭적기인위금류감HA기인공정역묘적연구전정료기출。
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.