[转移贴]我国科研人员克隆出抗狂犬病毒新抗体

cao1976

原贴由503814 发表于 2011-4-6 21:17
http://biosky.haotui.com/thread-21313-1-4.html

能有效阻止狂犬病毒感染；在狂犬病预防中具有潜在应用前景

来自南京军区军事医学研究所和南京医科大学的科研人员采用克隆方法，制备出一株具有中和活性的抗狂犬病毒的基因工程中和新型抗体（Fab）。经动物实验证实，该抗体联合疫苗能有效阻止狂犬病毒感染，在狂犬病预防中具有潜在的应用前景。该论文发表在最新一期《中国药理学报》上。

狂犬病是由狂犬病毒引起的人畜共患急性传染病，病死率极高，表现为极度神经兴奋乃至狂暴，继之局部或全身麻痹而死亡。狂犬病发病后进展速度快，至多10天内死亡。据联合国世界卫生组织的相关材料显示，每年约有5万人和数以百万计的野生动物死于狂犬病，中国因狂犬病而死亡的人数在法定传染病中已跃居第2位。

联合应用抗狂犬病毒免疫球蛋白和狂犬病疫苗是狂犬病三级感染预防的主要措施。目前临床使用的抗狂犬病毒免疫球蛋白，主要来自狂犬病疫苗免疫人或马的抗狂犬病毒血清，抗体的产量受到限制且存在一定的安全隐患。基因工程抗体技术的完善与发展，为制备具备高亲和力的治疗性人源抗体提供新途径。人源抗体由于具有较低的免疫原性和较好的组织穿透性，使其能够更好地应用于疾病的临床治疗。

据悉，本研究是人源抗狂犬病毒治疗性中和抗体研究中的一部分，研究小组从人源免疫型抗狂犬病毒抗体库中筛选出的一株针对狂犬病毒糖蛋白的单链抗体，克隆出可变区基因，运用重叠延伸拼接的方法，拼接成Fab基因，并经过小鼠动物实验。体外研究及小鼠狂犬病毒感染后预防研究证实，抗体联合疫苗能够有效阻止狂犬病毒的感染，对狂犬病毒感染后的小鼠具有预防保护作用。

Acta Pharmacologica Sinica (2011) 32: 329–337; doi: 10.1038/aps.2010.209; published online 31 Jan 2011

Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

Chen LI1, 3, 4, Feng ZHANG1, 3, Hong LIN1, Zhong-can WANG2, Xin-jian LIU1, Zhen-qing FENG1, 3, Jin ZHU1, 2, *, Xiao-hong GUAN1, *

1Key Laboratory of Antibody Technique of Ministry of Health, Nanjing Medical University, Nanjing 210029, China; 2Huadong Medical Institute of Biotechniques, Nanjing 210002, China; 3Department of Pathology, Nanjing Medical University, Nanjing 210029, China; 4Department of Pathology, the Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China


Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.
Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS.  The recombinant vector was then transformed into E coli Top10F′ for expression and purification.  The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.

Results: A recombinant vector was constructed.  The Fab was expressed in soluble form in E coli Top10F′.  Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP).  The neutralizing antibody titer of Fab was 10.26 IU/mL.  The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).

Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

Keywords: rabies; Fab engineered antibody; neutralizing antibodies