基于層析技術(shù)探究金環(huán)蛇蛇毒多克隆抗體F(ab′)₂的制備工藝

摘 要： 為制備高純度馬源金環(huán)蛇毒多克隆抗體F（ab′）₂，以金環(huán)蛇毒高免馬血漿為原料，采用Protein G親和層析捕獲馬血漿中的IgG，經(jīng)胃蛋白酶酶解后制得F（ab′）₂，分別通過(guò)親和層析、凝膠過(guò)濾層析和混合模式層析對(duì)F（ab′）₂進(jìn)行精制純化，并以間接ELISA法測(cè)定F（ab′）₂對(duì)金環(huán)蛇毒的中和活性。結(jié)果表明：Protein G親和層析捕獲的IgG純度為83.6%，較傳統(tǒng)的鹽析純化法有明顯提高；在3種層析精制方法中，通過(guò)凝膠過(guò)濾層析精制的F（ab′）₂純度最高，為89.3%，遠(yuǎn)高于《中華人民共和國(guó)藥典》標(biāo)準(zhǔn)（60%以上），且收率為56.0%；間接ELISA法測(cè)定結(jié)果顯示，F(xiàn)（ab′）₂的效價(jià)高于1∶4096。該研究為馬源抗毒素多克隆抗體F（ab′）₂的工業(yè)化制備提供了工藝參考。

關(guān)鍵詞：金環(huán)蛇；多克隆抗體；胃蛋白酶；F（ab′）₂片段；層析純化

中圖分類號(hào)：R392-33; R392.7

文獻(xiàn)標(biāo)志碼：A

文章編號(hào)：1673-3851 （2025） 03-0276-07

引文格式：李爽，王煒杰，趙偉睿，等. 基于層析技術(shù)探究金環(huán)蛇蛇毒多克隆抗體F（ab′）₂的制備工藝［J］. 浙江理工大學(xué)學(xué)報(bào)（自然科學(xué)），2025，53（2）：276-282.

Reference Format： LI Shuang，WANG Weijie，ZHAO Weirui，et al. A study on the preparation technology of F（ab′）₂ fragments from polyclonal antibodies against Bungarus fasciatus venoms by chromatography［J］. Journal of Zhejiang Sci-Tech University，2025，53（2）：276-282.

A study on the preparation technology of F（ab′）₂ fragments from polyclonal

antibodies against Bungarus fasciatus venoms by chromatography

LI Shuang¹，WANG Weijie²，ZHAO Weirui²，HU Sheng²，WANG Lei³，F(xiàn)AN Quanshui³，L Changjiang⁴，HUANG Jun⁴，MEI Lehe^1，2，5

（1.College of Chemical and Biological Engineering， Zhejiang University， Hangzhou 310027， China;

2.School of Biological and Chemical Engineering， NingboTech University， Ningbo 315100， China;

3.Zhejiang Jianbo Biotechnology Co.， Ltd.， Ningbo 315602， China; 4.School of Biological and Chemical

Engineering， Zhejiang University of Science and Technology， Hangzhou 310023， China;

5.Jinhua Advanced Research Institute， Jinhua 321019， China）

Abstract：To prepare high-purity horse-derived F（ab′）₂ fragments of polyclonal antibodies against Bungarus fasciatus venoms， IgG was captured from hyperimmuned horse plasma with Bungarus fasciatus venoms as the raw material by Protein G affinity chromatography， and then the prepared IgG was enzymatically cleaved by pepsin into F（ab′）₂ fragments， which were purified by affinity chromatography， gel filtration chromatography， and mixed-mode chromatography， respectively. The neutralizing activity of the prepared F（ab′）₂ against Bungarus fasciatus venoms was determined by indirect ELISA. The results showed that IgG captured by Protein G affinity chromatography attained a purity of 83.6%， which was significantly higher than that of the traditional salting-out method. Among the three chromatographic refinement methods we tested， gel filtration chromatography refined F（ab′）₂ had the highest purity of 89.3%， which was much higher than the standard of Pharmacopoeia of the People’s of Republic of China （more than 60%）， and the recovery was 56.0%. The titer of the F（ab′）₂ was higher than 1：4096 by indirect ELISA. The study provides reference for the industrialized preparation of horse-derived antitoxin polyclonal antibody F（ab′）₂.

Key words：Bungarus fasciatus; polyclonal antibody; pepsin; F（ab′）₂ fragments; chromatographic purification

0 引 言

金環(huán)蛇（Bungarus fasciatus）屬眼鏡蛇科環(huán)蛇屬，是以神經(jīng)毒素為主、兼有心臟毒素的劇毒蛇類，主要分布在中國(guó)南部地區(qū)，常于清晨或夜間出沒于山溝或草叢中，嚴(yán)重威脅居民和野外作業(yè)人員的生命安全^［1］。抗蛇毒血清是治療蛇毒咬傷的特效藥，但目前中國(guó)尚無(wú)商業(yè)化的抗金環(huán)蛇毒血清，臨床上使用抗銀環(huán)蛇毒血清來(lái)治療金環(huán)蛇咬傷^［2］。該法起到了一定的治療效果，但相比金環(huán)蛇毒本身免疫制備的抗血清，異種蛇毒免疫制備的抗血清對(duì)金環(huán)蛇毒的中和效價(jià)低。此外，采用該法后，注入人體的抗血清蛋白總量也隨之升高，易導(dǎo)致體內(nèi)發(fā)生血清不良反應(yīng)，給金環(huán)蛇咬傷患者的有效救治帶來(lái)極大隱患。……