利用报告基因的人7型腺病毒中和抗体检测方法的建立  

Establishment of a Reporter Gene Assay for Detection of Human Adenovirus Type 7 Neutralizing Antibody

作　　者：李建华 王步森 吴诗坡 王玉东 赵拯浩 侯利华 LI Jian-Hua;WANG Bu-Sen;WU Shi-Po;WANG Yu-Dong;ZHAO Zheng-Hao;HOU Li-Hua(Beijing Institue of Biotechnology,Beijing 100071,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2018年第6期770-777,共8页Letters in Biotechnology

摘　　要：目的:构建表达萤光素酶报告基因的重组人7型腺病毒(HAdV7)Ad7-dE1-dE3-Luc(Ad7-Luc)病毒,用于检测人群中HAdV7的中和抗体水平。方法:利用同源重组方法构建E1区和E3区部分缺失,并在E1区嵌入萤光素酶基因表达框的pAd7-Luc重组质粒,转染HEK-293细胞,包装出能够表达萤火虫萤光素酶的Ad7载体重组病毒(Ad7-Luc);选取HAdV7阳性血清样本对新建立的报告基因检测方法的准确性和稳定性进行验证;最后,利用该方法对北京地区60份血清样本进行HAdV7中和抗体检测。结果:获得能够表达萤火虫萤光素酶的Ad7-Luc重组病毒,病毒滴度可达4.85×10~8IFU/mL,Ad7-Luc检测法与传统细胞病变效应(CPE)法具有很好的一致性(R^2=0.8612,P<0.0001),批内和批间差异分别在10%和20%以内。北京地区60例血清样本的检测结果显示,HAdV7血清阳性率约为60%(36/60),明显低于HAdV5血清阳性率75%(45/60),75%的HAdV7阳性血清中和抗体滴度集中于低滴度水平(12～200)。结论:与传统的细胞病变方法相比,HAdV7中和抗体报告基因检测方法更快速、灵敏、准确,适用于人群中HAdV7血清流行病学调查及其疫苗的中和抗体研究。Objective:A recombinant human adenovirus type7(HAdV7)expressing the luciferase reporter gene(Ad7-dE1-dE3-Luc,Ad7-Luc)was constructed to detect neutralizing antibodies of HAdV7in the population in Beijing.Methods:The pAd7-Luc recombinant plasmid was constructed by partially deleting E1and E3region,and inserting luciferase gene in E1region.A new HAdV7neutralization assay was established based on the Ad7-Luc recombinant virus,which was rescued by transfecting linearized pAd7-Luc plasmids into HEK-293cells,and its accuracy and stability were evaluated by luciferase-based method using several HAdV7-positive serum samples.Finally,60serum samples from Beijing were examined.Results:The Ad7-Luc recombinant virus expressing luciferase was constructed,and the virus titer was up to4.85×10^8IFU/mL.There was a good correlation in serum titers measured by Ad7-Luc method and the traditional cytopathic effect(CPE)assay,intra-and inter-batch differences can be within10%and20%respectively.The HAdV7seropositive rate of60serums in Beijing was60%(36/60),significantly lower than that of Ad575%(45/60),and the neutralizing antibody titers of75%of Ad7-positive serums were at low levels(12~200).Conclusion:Compared with the traditional CPE method,the HAdV7neutralizing antibody detection method based on the luciferase expression is more rapid,sensitive and accurate,and is suitable for HAdV7serum epidemiological investigation and neutralizing antibody assay for HAdV7vaccine.

关 键 词：人7型腺病毒 萤光素酶报告基因 中和抗体 血清流行病学调查 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]