华中科技大学学报（医学版）
華中科技大學學報（醫學版）
화중과기대학학보（의학판）
ACTA UNIVERSITATIS MEDICINAE TONGJI
2015年
4期
390-394
,共5页
噬菌体展示%单链抗体%血管细胞黏附分子-1%重组抗体
噬菌體展示%單鏈抗體%血管細胞黏附分子-1%重組抗體
서균체전시%단련항체%혈관세포점부분자-1%중조항체
phage display%single chain variable fragment antibody%vascular cell adhesion molecule-1%recombinant antibody
目的：从噬菌体重组抗体库中筛选获得靶向血管细胞黏附分子‐1（Vcam‐1）的单链抗体，纯化浓缩后进行亲和性鉴定，并与单克隆抗体效价进行比较。方法扩增Vcam‐1基因克隆质粒并转入真核细胞中表达获得Vcam‐1抗原蛋白，纯化后包被免疫管，通过4轮压力逐渐增大的“吸附‐洗脱‐扩增”过程筛选获得阳性克隆。对阳性克隆进行ELISA检测，选取效价高的克隆送予测序并转入大肠埃希菌进行表达，认定高表达的样品为最终的阳性克隆。将该阳性克隆转染入感受态细胞表达单链抗体，纯化后经ELISA检测并评价其抗原亲和性。结果真核细胞表达的Vcam‐1抗原蛋白的分子量为85～90 kD。以Vcam‐1抗原蛋白为免疫原筛选4轮所得的阳性克隆进行单噬菌体ELISA检测，从检测结果中选取9个效价高的克隆经基因测序共获得3个序列，其中1个序列对应的克隆高表达。表达的单链抗体分子量约30 kD ，ELISA检测其对Vcam‐1抗原蛋白有较高亲和性，效价较单克隆抗体低。结论利用噬菌体展示技术获得了靶向Vcam‐1的单链抗体，为随后的诊断和治疗应用奠定了基础。
目的：從噬菌體重組抗體庫中篩選穫得靶嚮血管細胞黏附分子‐1（Vcam‐1）的單鏈抗體，純化濃縮後進行親和性鑒定，併與單剋隆抗體效價進行比較。方法擴增Vcam‐1基因剋隆質粒併轉入真覈細胞中錶達穫得Vcam‐1抗原蛋白，純化後包被免疫管，通過4輪壓力逐漸增大的“吸附‐洗脫‐擴增”過程篩選穫得暘性剋隆。對暘性剋隆進行ELISA檢測，選取效價高的剋隆送予測序併轉入大腸埃希菌進行錶達，認定高錶達的樣品為最終的暘性剋隆。將該暘性剋隆轉染入感受態細胞錶達單鏈抗體，純化後經ELISA檢測併評價其抗原親和性。結果真覈細胞錶達的Vcam‐1抗原蛋白的分子量為85～90 kD。以Vcam‐1抗原蛋白為免疫原篩選4輪所得的暘性剋隆進行單噬菌體ELISA檢測，從檢測結果中選取9箇效價高的剋隆經基因測序共穫得3箇序列，其中1箇序列對應的剋隆高錶達。錶達的單鏈抗體分子量約30 kD ，ELISA檢測其對Vcam‐1抗原蛋白有較高親和性，效價較單剋隆抗體低。結論利用噬菌體展示技術穫得瞭靶嚮Vcam‐1的單鏈抗體，為隨後的診斷和治療應用奠定瞭基礎。
목적：종서균체중조항체고중사선획득파향혈관세포점부분자‐1（Vcam‐1）적단련항체，순화농축후진행친화성감정，병여단극륭항체효개진행비교。방법확증Vcam‐1기인극륭질립병전입진핵세포중표체획득Vcam‐1항원단백，순화후포피면역관，통과4륜압력축점증대적“흡부‐세탈‐확증”과정사선획득양성극륭。대양성극륭진행ELISA검측，선취효개고적극륭송여측서병전입대장애희균진행표체，인정고표체적양품위최종적양성극륭。장해양성극륭전염입감수태세포표체단련항체，순화후경ELISA검측병평개기항원친화성。결과진핵세포표체적Vcam‐1항원단백적분자량위85～90 kD。이Vcam‐1항원단백위면역원사선4륜소득적양성극륭진행단서균체ELISA검측，종검측결과중선취9개효개고적극륭경기인측서공획득3개서렬，기중1개서렬대응적극륭고표체。표체적단련항체분자량약30 kD ，ELISA검측기대Vcam‐1항원단백유교고친화성，효개교단극륭항체저。결론이용서균체전시기술획득료파향Vcam‐1적단련항체，위수후적진단화치료응용전정료기출。
Objective To screen out single chain variable fragment antibody (scFv)specific to vascular cell adhesion mole‐cule 1(Vcam‐1)from phage recombinant antibody library ,and to evaluate its activity and compare its activity with full‐length monoclonal antibody.Methods Amplification of Vcam‐1 was performed by PCR and Vcam‐1 gene plasmid was transferred into eukaryotic cells to express Vcam‐1 antigen protein.Immune cuvette was coated with purified Vcam‐1 antigen ,and the positive clones were screened out by 4 rounds of “adhesion‐elution‐proliferation” process with gradually increasing pressure.The posi‐tive clones were tested by ELISA method and high titer clones were chosen for gene sequencing.Then the high‐titer clones were transferred into E.coli ,and the clone with the highest expression was regarded as the final requisite one.Competent cells were infected by the final requisite clone and scFv was expressed.After purification ,the activity of scFv was tested by ELISA and its affinity was evaluated.Results Molecular weight of Vcam‐1 antigen protein was 85-90 kD.Positive clones were screened out by taking Vcam‐1 protein as the antigen ,and 9 high titer clones were obtained by single phage ELISA.Gene sequencing of these clones was carried out and 3 sequences were obtained ,1 of which got the highest expression.Molecular weight of the expressed scFv was about 30 kD.The scFv got high affinity to Vcam‐1 antigen according to ELISA ,in spite of its lower activity than full‐length monoclonal antibody.Conclusion scFv antibody specific to Vcam‐1 was successfully obtained from phage display librar‐y ,which laid the foundation of subsequent in vivo diagnosis and therapy.
