CAJ | 학술논문

目的从噬菌体抗体库中筛选抗CCR7单链融合抗体,并对其生物学特性进行初步检测.方法 PCR检测大肠杆菌中ScFv基因插入率;琼脂糖凝胶电泳鉴定Sfi I和Not I双酶切质粒的结果;分别以乳腺癌细胞及CCR7多肽片段为靶抗原对抗体库进行4轮和3轮筛选富集.将阳性克隆转化E.coli HB2151进行可溶表达.抗体亲和层析纯化后,经Western blot鉴定,通过ELISA法检测可溶性ScFv抗体的免疫活性.免疫细胞化学和放射免疫显像鉴定scFv抗体与乳腺癌细胞结合的特异性.结果 ScFv基因插入率为90%(18/20),双酶切鉴定检测到目的条带.经4轮细胞筛选,3轮抗原筛选抗CCR7抗原的噬菌体抗体得到了明显富集,在E.coli HB2151中实现可溶表达.Western blot结果显示获得抗体相对分子质量为34 ku左右.免疫细胞化学检测与放射免疫显像均证实单链抗体与表达CCR7的乳腺癌细胞MDA-MB-435s特异性结合.结论成功从噬菌体抗体库中筛选获得具有较高特异性的抗CCR7单链抗体.抗体在体内体外均与肿瘤细胞表达特异抗原结合.
목적 종서균체항체고중사선항CCR7단련융합항체,병대기생물학특성진행초보검측.방법 PCR검측대장간균중ScFv기인삽입솔;경지당응효전영감정Sfi I화Not I쌍매절질립적결과;분별이유선암세포급CCR7다태편단위파항원대항체고진행4륜화3륜사선부집.장양성극륭전화E.coli HB2151진행가용표체.항체친화층석순화후,경Western blot감정,통과ELISA법검측가용성ScFv항체적면역활성.면역세포화학화방사면역현상감정scFv항체여유선암세포결합적특이성.결과 ScFv기인삽입솔위90%(18/20),쌍매절감정검측도목적 조대.경4륜세포사선,3륜항원사선항CCR7항원적서균체항체득도료명현부집,재E.coli HB2151중실현가용표체.Western blot결과현시획득항체상대분자질량위34 ku좌우.면역세포화학검측여방사면역현상균증실단련항체여표체CCR7적유선암세포MDA-MB-435s특이성결합.결론 성공종서균체항체고중사선획득구유교고특이성적항CCR7단련항체.항체재체내체외균여종류세포표체특이항원결합.
Aim To screen and identify anti-CCR7 single chain fragments variable(scFv)from lager phage library and to detect the scFv efficiency.Methods The insert ratio of ScFv antibodies library was identified by PCR.The products digested by Sfi I/Not I double enzyme.Panning against breast cancer cell and CCR7 were performed four and three rounds respectively.Positive clones were transformed to E.coli HB2151, and their dissolvability was assayed.The soluble scFv was purified by affinity chromatography, and its relative molecular mass was determined by Western blot.The ELISA assay was used to identify the immunocompetence of the antibody.Immunocytochemical staining and radioimmunoimaging were employed to determine the affinities of scFv binding with CCR7 in cell line and in nude mice.Results The insert ratio of ScFv gene was 90%(18/20), enzyme digest reaction showed the aim products on 1% agarose gel.ScFvs were obviously enriched after 7 round panning.Western blot result showed soluble scFv's molecular mass was about 34 KD.ELISA analysis showed dissolved antibody had high immunocompetence to MDA-MB-435 s cells.Immunocytochemical staining and radioimmunoimaging indicated that ScFvs were bound efficiently to MDA-MB-435 s cells which expressed CCR7.Conclusions ScFvs against CCR7 are successfully acquired by screening the phage antibody library.The soluble ScFvs have high affinity and specifical binding to human breast cancer cells.