组织因子人—鼠嵌合抗体制备的研究

发布时间：2018-05-26 15:39

本文选题：TF + 凝血因子Ⅲ　；参考：《山西大学》2010年硕士论文

【摘要】： 组织因子(tissue factor, TF)是相对分子量为47 kD的跨膜糖蛋白,在调节凝血功能方面有重要的作用,近年来研究发现TF参与动脉粥样硬化的发生发展、血管生成和肿瘤生长等非凝血功能。利用TF单克隆抗体的导向性和特异性,抑制TF的活性,从而抑制肿瘤的生长。由于鼠源性抗体药物对人类具有免疫原性,其广泛的临床应用受到极大限制,而采用基因工程技术对鼠源性抗体进行人源化改造是治疗用抗体的重要手段。因此,本文在自行研制的抗人TF鼠源性单克隆抗体的基础上,利用基因工程抗体技术,成功构建了抗人TF人-鼠嵌合抗体基因的真核共表达质粒。将其转染CHO-K1细胞,筛选获得能分泌表达TF嵌合抗体的CHO稳定细胞株(JJ3),经Protein A亲和层析柱纯化获得高纯度的抗TF嵌合抗体,体外分析该抗体具有抗凝血活性和对K562和HL-60癌细胞生长具有明显的抑制作用,为TF抗体治疗的临床应用奠定基础。首先利用RNA提取试剂盒,从分泌TF鼠源性抗体的杂交瘤细胞(mTA)中提取总RNA,以鼠源性TF单克隆抗体重链可变区(VH)及轻链可变区(VL)基因序列设计特异性引物,采用RT-PCR获得重链和轻链可变区目的基因。将目的基因分别与pCN40表达载体连接,构建成重组质粒pCN40-VL-VH。经AgeⅠ、BstBⅠ和HpaⅠ、EcoRⅠ分别对重组质粒酶切鉴定和琼脂糖凝胶电泳分析,两条目的基因片段大小约为300 bp,与理论值(321 bp、351 bp)相吻合,测序分析其序列与目的基因序列一致,表明已成功构建了抗人TF人-鼠嵌合抗体重组质粒pCN40-VL-VH。其次,以中国仓鼠卵巢细胞(CHO-K1)为宿主细胞,采用中量无内毒素提取质粒试剂盒提取重组质粒pCN40-VL-VH,阳离子脂质体方法转染CHO-K1细胞,经G418硫酸盐(800μg/mL)筛选,并通过有限稀释法克隆化培养,培养上清经间接ELISA检测。对筛选的分泌抗体阳性细胞株培养上清经间接ELISA、Western blot方法分析,获得了转染重组质粒pCN40-VL-VH的CHO细胞株(JJ3),并能稳定地分泌抗人TF人-鼠嵌合抗体。利用rProtein A亲和层析柱纯化该细胞培养上清中的抗体,纯化后的产品经紫外分光光度计定量分析抗体产量约为50 mg/L。经SDS-PAGE分析：纯化嵌合抗体的重链和轻链的分子量分别约为50 kD和26 kD,纯度达到97%以上。最后,选择K562、HL-60、LOVO、BEL-7402人肿瘤细胞株,采用MTT法,体外研究了该纯化抗体对肿瘤细胞增殖的影响。结果表明：在一定浓度范围内,该抗体能够明显地抑制K562和HL-60细胞的生长,最大抑制率分别为35%和64.9%,且呈剂量效应关系,而对LOVO和BEL-7402细胞生长没有明显的影响。不同浓度的嵌合抗体与27.4μg/mL的TF温育1.5 h,经脂化和凝血酶原时间测定,表明随着嵌合抗体浓度的升高,与对照组相比,正常人血浆的PT显著延长(P0.01)。上述结果表明：本研究构建了抗人TF人-鼠嵌合抗体重组质粒pCN40-VL-VH,转染宿主细胞并获得能够稳定表达抗人TF人-鼠嵌合抗体的CHO-K1细胞株(JJ3),经亲和层析获得纯度达97%以上的抗人TF人-鼠嵌合抗体,体外实验表明该抗体具有抗凝活性和有效地抑制K562和HL-60细胞增殖的活性。
[Abstract]:Tissue factor ( TF ) is a transmembrane glycoprotein with a relative molecular weight of 47 kD . It plays an important role in regulating the coagulation function . In recent years , it has been found that TF is involved in the development of atherosclerosis , angiogenesis and tumor growth .

The recombinant plasmid pCN40 - VL - VH was amplified by RT - PCR . The recombinant plasmid pCN40 - VL - VH was amplified by RT - PCR . The recombinant plasmid pCN40 - VL - VH was constructed by RT - PCR . The recombinant plasmid pCN40 - VL - VH was constructed by sequencing and sequencing . The recombinant plasmid pCN40 - VL - VH was constructed successfully .

The recombinant plasmid pCN40 - VL - VH was extracted from Chinese hamster ovary cells ( CHO - K1 ) . The recombinant plasmid pCN40 - VL - VH was extracted with a medium dose of the plasmid pCN40 - VL - VH . The recombinant plasmid pCN40 - VL - VH was screened by indirect ELISA . The purified product was purified by indirect ELISA . The purified product was purified by indirect ELISA . The purified product was analyzed by SDS - PAGE . The molecular weight of the heavy chain and light chain of purified chimeric antibody was about 50 kD and 26 kD , respectively , and the purity was above 97 % .

Finally , the effects of the purified antibody on the proliferation of tumor cells were studied by MTT assay . The results showed that the growth of K562 and HL - 60 cells was significantly inhibited by MTT assay . The maximal inhibitory rates were 35 % and 64.9 % , respectively , and there was no significant effect on the growth of LOVO and BEL - 7402 cells .

The results showed that the recombinant plasmid pCN40 - VL - VH of anti - human TF human - mouse chimeric antibody was constructed and the CHO - K1 cell line ( JJ3 ) capable of stably expressing anti - human TF human - mouse chimeric antibody was constructed . The anti - human TF human - mouse chimeric antibody with purity of over 97 % was obtained by affinity chromatography . The in vitro experiment showed that the anti - human TF human - mouse chimeric antibody had anticoagulant activity and effectively inhibited the proliferation of K562 and HL - 60 cells .
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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