來自南京*軍事醫(yī)學(xué)研究所和南京醫(yī)科大學(xué)的科研人員采用克隆方法，制備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體（Fab）。經(jīng)動(dòng)物實(shí)驗(yàn)證實(shí)，該抗體聯(lián)合疫苗能有效阻止狂犬病毒感染，在狂犬病預(yù)防中具有潛在的應(yīng)用前景。該論文發(fā)表在一期《中國(guó)藥理學(xué)報(bào)》上。
狂犬病是由狂犬病毒引起的人畜共患急性傳染病，病死率*，表現(xiàn)為極度神經(jīng)興奮乃至狂暴，繼之局部或全身麻痹而死亡?？袢“l(fā)病后進(jìn)展速度快，至多10天內(nèi)死亡。據(jù)聯(lián)合國(guó)世界衛(wèi)生組織的相關(guān)材料顯示，每年約有5萬人和數(shù)以百萬計(jì)的野生動(dòng)物死于狂犬病，中國(guó)因狂犬病而死亡的人數(shù)在法定傳染病中已躍居第2位。
聯(lián)合應(yīng)用抗狂犬病毒免疫球蛋白和狂犬病疫苗是狂犬病三級(jí)感染預(yù)防的主要措施。目前臨床使用的抗狂犬病毒免疫球蛋白，主要來自狂犬病疫苗免疫人或馬的抗狂犬病毒血清，抗體的產(chǎn)量受到限制且存在一定的安全隱患。基因工程抗體技術(shù)的完善與發(fā)展，為制備具備高親和力的治療性人源抗體提供新途徑。人源抗體由于具有較低的免疫原性和較好的組織穿透性，使其能夠更好地應(yīng)用于疾病的臨床治療。
據(jù)悉，本研究是人源抗狂犬病毒治療性中和抗體研究中的一部分，研究小組從人源免疫型抗狂犬病毒抗體庫中篩選出的一株針對(duì)狂犬病毒糖蛋白的單鏈抗體，克隆出可變區(qū)基因，運(yùn)用重疊延伸拼接的方法，拼接成Fab基因，并經(jīng)過小鼠動(dòng)物實(shí)驗(yàn)。體外研究及小鼠狂犬病毒感染后預(yù)防研究證實(shí)，抗體聯(lián)合疫苗能夠有效阻止狂犬病毒的感染，對(duì)狂犬病毒感染后的小鼠具有預(yù)防保護(hù)作用。
原文出處：
Acta Pharmacologica Sinica （2011） 32: 329–337; doi: 10.1038/aps.2010.209
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, *
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 *0F′ 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 *0F′. 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
