1、卵巢癌抗独特型单链抗体6B11ScFv/mGM-CSF融合蛋白的构建、表达及活性测定【摘要】目的构建卵巢癌抗独特型单链抗体6B11ScFv和鼠GM-CSF融合蛋白(6B11mGM)，以观察其在动物体内诱导的特异性免疫反应，为卵巢癌抗独特型疫苗应用于临床提供依据。方法用DNA重组技术，将mGM-CSF连于单链抗体6B11ScFv羧基末端，构建重组质粒pET30-6B11mGM，转化大肠杆菌BL21(DE3)，IPTG诱导，以包涵体形式获高效表达，超声破碎细菌细胞获得包涵体，用8mol.L-1尿素溶解包涵体后直接稀释复性，SDS-PAGE分析蛋白纯度，ELISA分析技术和细胞增殖实验测定融合蛋白的
2、抗体和细胞因子活性。结果复性蛋白纯度达90%以上。采用氧化型谷胱甘肽(GSSG)浓度为1mmol.L-1，还原型谷胱甘肽(GSH)浓度为5mmol.L-1，10复性48h，复性率达36%。表达的融合蛋白分别能与COC166-9单抗和大鼠抗小鼠GM-CSF单抗特异结合，并能刺激mGM-CSF依赖株NFS-60细胞增殖。结论以包涵体表达的融合蛋白6B11mGM保留了两种蛋白的活性，为研究融合蛋白在体内的免疫功能提供了基础。【关键词】卵巢癌；重组融合蛋白；抗体；抗独特型；粒细胞-巨噬细胞集落刺激因子；包涵体【中分类号】R392.11【文献标识码】A【文章编号】0529-1356(2000)03-22
3、6 THE CONSTRUCTION， EXPRESSION AND ACTIVITY EXAMINATIONSOF 6B11ScFvMURINE GM-CSF FUSION PROTEINLIU Bei,CUI Heng?, FENG Jie, YE Xue, LI YI, CAO Shan-jin, GE Hua, FU Tian-yun, YAO Yu, QIAN He-nian(Gynecologic Oncology Center, Peoples Hospital, Beijing Medical University, Beijing100044, China)【Abstract
4、】ObjectiveTo construct expression vector which expresses fusion protein of antiidiotypic single chain antibody 6B11ScFv and murine GM-CSF(6B11mGM) for observing its possible immune reactions in vivo. MethodsUsing DNA recombinant techniques, the murine GM-CSF cDNA gene was recombined to 6B11ScFv and
5、they were cloned into expression vector pET-30a(+) to produce insoluble protein. Inclusion bodies collected after the breakage of bacteria through sonication were subjected to repeatedly washing. Inclusion bodies solubilized in the resence of 8 mol.L-1 urea were diluted with renaturation solutions s
6、o that folding process could be initiated. The purity was examined by SDS-PAGE, ELISA and cell proliferation assay were used to determine the activities of fusion protein. Results6B11mGM fusion proteins were obtained with a purity of over 90%. The optimum conditions were 1mmol.L-1 and 5 mmol.L-1 for
7、 the concentrations of GSSG and GSH, respectively, with 48 hours at 10 as renatured time. Fusion protein could specifically react with the primary anti-ovarian carcinoma monoclonal antibody(COC166-9) and rat anti-mouse GM-CSF monoclonal antibody, respectively, and fusion proteins could also stimulat
8、e murine GM-CSF dependent cell line NFS-60 cells to proliferate. ConclusionFusion proteins 6B11mGM expressed as inclusion bodies can keep the activity of both the proteins and provid foundation to research the immunoreactions in vivo.【Key words】Ovarian carcinoma; Recombinant fusion proteins; Antibod
9、ies, Anti-idiotypic; Granulocyte-macrophage colony-stimulating factor; Inclusion bodies本中心曾用卵巢上皮癌可溶性抗原OC166-9免疫BALBc小鼠，制备了单克隆抗体COC166-9。经免疫组织化学染色证实该抗体对卵巢上皮癌具有较高的特异性1。继而用COC166-9单抗免疫小鼠制备了内影像型抗独特型抗体6B11。经免疫竞争抑制试验证实，该抗体具有模拟卵巢上皮癌初始抗原OC166-9的作用2，可在体内外诱导产生特异的抗卵巢癌免疫反应3，4。为了降低鼠源性抗体反复在体内应用时可能引起的副作用，本中心对卵巢癌抗独
10、特型抗体6B11进行改造，构建了单链抗体6B11ScFv5。改型后的抗体，虽降低了鼠源性，但同时因分子量减少，免疫原性也随之下降，应用时必须与匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)等偶联并加用佐剂才能引起有效的免疫反应6，而现在常用佐剂往往对人体有害。因此，有必要寻求一种新的佐剂。最近研究表明粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)等细胞因子与抗原构成的融合蛋白，可增加抗原的免疫原性7。为提高单链抗体6B11ScFv的免疫原性，使其能做为卵巢癌抗独特型疫苗，
11、本中心已构建成功6B11ScFv与人GM-CSF融合蛋白(6B11GM)，并且体外实验已证实在抗原呈递细胞(antigen presenting cell,APC)和树突状细胞(dendritic cell,DC)存在情况下，可诱导T细胞增殖，同时APC对6B11GM摄取明显高于单用6B118。但由于目前缺乏合适的动物模型，使抗独特型疫苗的效果和机制难以得到体内实验的验证。因此，本研究构建6B11ScFv与鼠GM-CSF融合蛋白(6B11mGM)，目的在于进行体内外实验，为卵巢癌抗独特型疫苗6B11GM应用于临床打下基础。材料和方法1.质粒、细胞系质粒pL6B11ScFv-hGM-CSF由本中
12、心构建。质粒pET30a(+)购自Novagen公司。质粒pTC-GM.2内含murine GM-CSF cDNA，由美国斯坦福大学Levy教授惠赠。pGEM-T Vector购自Prome-ga公司。鼠GM-CSF依赖株NFS-60细胞购自北京医科大学分子免疫学室。宿主菌E.coli BL21(DE3)购自Novagen公司。2.抗原、抗体和细胞因子卵巢浆液性乳头状癌抗原OC166-9由本中心制备。HRP-COC166-9抗体、6B11单克隆抗体均为本室制备。大鼠抗小鼠GM-CSF单抗及重组murine GM-CSF购自美国R and D System。HRP-羊抗大鼠IgG购自Santa
13、Cluz公司。3.试剂Tag酶、T4DNA连接酶及各种DNA限制性内切酶均购自美国Promega公司。MTT等生化试剂购自北京天象人公司和北京化学试剂公司。4.鼠GM-CSF的PCR克隆鼠GM-CSF位于pTC-GM.2质粒中，由于缺乏合适的酶切位点，依据融合蛋白6B11mGM的构建策略(13)。利用PCR克隆技术，在mGM-CSF上、下游引物中分别引入匹配的酶(SpeI、EcoRI)切点。上游引物：5-TATACTAGTGCACCCACCC-GCTCACCCAT-3；下游引物：5-GGGGAATTC-TCATTTTTGGACTGGTTT-3。为避免PCR可能产生的突变，将mGM-CSF的PC
14、R产物，克隆入测序载体pGEM-T Vector，构建成pGEM-TmGM-CSF。经Sanger双脱氧末端终止法测序，证实所克隆到的mGM-CSF与文献发表序列及Genebank登录序列完全一致9(1)。引物合成、测序工作均由赛百盛生物工程公司完成。5.6B11mGM融合蛋白表达质粒的构建为获得融合蛋白的高表达，我们选择了原核高表达质粒载体pET30a(+)。首先将6B11ScFv-hGM-CSF以合适的酶切位点自pL6B11ScFv-hGM-CSF中完整切下，克隆到pET30a(+)之中，构建成pET306B11ScFv-hGM-CSF(2)。之后将1.4中成功克隆到的mGM-CSF，自p
15、GEM-TmGM-CSF中切下，置换pET306B11ScFv-hGM-CSF中的hGM-CSF，从而构建成功6B11GM融合蛋白的高表达载体pET306B11ScFv-mGM-CSF(3)。连接产物转化DH5受体菌，内切酶酶切鉴定阳性克隆。将筛选出的pET306B11ScFv-mGM-SCF阳性克隆，转化BL21(DE3)受体菌，进行诱导表达。6.6B11mGM融合蛋白的表达及包涵体处理将含有pET306B11ScFv-mGM-SCF的BL21(DE3)受体菌经37培养，以0.4mmol*L-1IPTG诱导表达4h，收获菌体。超声破碎细菌，释放包涵体。10 000r*min-1离心20min，弃上清。将沉淀物用3mol*L-1尿素，含0.2g*L-1TritonX-100反复洗涤处理，得到纯化的包涵体，称重。 1mGM-CSF的PCR克隆Fig.The cloning of mGM-CSF by PCR 2pET306B11ScFv-hGM-SCF构建策略示意Fig.2 The construction strategy of pET306B11ScFv-hGM-SCF 3pET306B11ScFv-mGM-CSF构建策略示意Fig.3 The con