【作者】 朱小玲；

【导师】 吴润； 张杰；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2006， 硕士

【摘要】 【目的】本研究拟采用秦川黄牛重组成熟朊蛋白(PrP)的纯化复性产物作为免疫原,应用淋巴细胞杂交瘤技术制备出抗牛成熟朊蛋白的单抗(mAb),为研制疯牛病免疫诊断试剂盒奠定基础。【方法】将本实验室保存的含有秦川黄牛成熟PrP基因的重组质粒pET30a-PrP (25~242aa)转入大肠杆菌E.coli JM109(DE3)中,IPTG诱导表达,SDS-PAGE分析鉴定表达产物。采用Ni-NTA亲和层析法纯化融合蛋白,透析法复性纯化的重组表达产物,考马斯亮蓝染色法测定蛋白浓度。以SAF70进口单抗为检测抗体,Western blot分析融合蛋白的免疫反应性和对蛋白酶K的敏感性。以纯化复性的牛成熟PrP融合蛋白作为免疫原,以含有pET30a(+)空载体的E.coli JM109(DE3)的菌体蛋白作为对照免疫原,分别与弗氏完全佐剂1:1混合乳化,按100μg/只剂量皮下3～4点注射8周龄清洁级BALB/c雌性小鼠,在第4周和第8周皮下注射同等剂量的以弗氏不完全佐剂乳化的免疫原,在第12周按150μg/只剂量直接腹腔注射PBS稀释的免疫原。3d后取PrP融合蛋白免疫小鼠脾细胞与SP2/0细胞在PEG1500作用下室温融合,HAT培养液选择培养,融合10～15d后取细胞上清液进行间接ELISA检测。纯化的牛成熟PrP融合蛋白和pET30a(+)空载体菌体蛋白分别按25μg/mL的工作浓度包被酶标板,融合蛋白免疫小鼠血清1:960和SAF70单抗1: 300作阳性对照,SP2/0细胞上清液1:1和对照免疫鼠血清1:960作阴性对照,加入辣根过氧化酶标记的山羊抗小鼠IgG,测定OD490nm吸收值。凡杂交瘤细胞液的OD490nm值大于阴性对照3倍以上者初步判定为阳性克隆。将阳性克隆N64杂交瘤作有限稀释,3次亚克隆后液氮冻存细胞并连续传代3个月检测杂交瘤分泌抗体的稳定性。将N64杂交瘤细胞按1×10~6腹腔注射经无菌液体石蜡预处理的BALB/c小鼠制备腹水单抗。以Protein A Agarose亲和层析法纯化腹水抗体,双抗体夹心法检测N64单抗的Ig类和亚类,间接ELISA法测定纯化的腹水抗体的效价和相对亲和力,Western blot分析N64单抗的特异性,采用基因片段表达作图法初步分析抗原表位。【结果】获得了能稳定分泌抗牛成熟朊蛋白的杂交瘤细胞株N64,分泌的抗体属于IgG2a,腹水效价为1×10~5,相对亲和力为0.2μg/mL。Western blot表明N64单抗具有较强的特异性,表位分析显示N64单抗仅与羧基端重组朊蛋白反应。更多还原

【Abstract】 Objective: To obtain monoclonal antibody (mAb) against mature prion proteins(PrP)of Qinchuan yellow cattle, the purified and refolded recombinant bovine mature prion proteins were regarded as immunogen, so that it will establish base for BSE immune diagnosis reagent kit.Method: Transforming recombinant plasmids(pET30a-PrP(25~242aa) contained Qinchuan yellow cattle PRNP and conserved in the lab into E. coli JM109(DE3), the expressed products were obtained from reombinant bacterium induced by IPTG and were identified correctly by SDS-PAGE analysis. Since then, the fusion proteins were purified by Ni-NTA immunoaffinity chromatograghy. The recombinant expression products were renatured by dialysis and their protein concentration were mensurated. As import mAb SAF70 for detect antibody, Western blot analysed reactiongenicity and proteinase K sensitivity of the fusion proteins. The purified and renatured fusion proteins of bovine mature PrP as immunogene (positive immuogene) and proteins of E. Coli JM09 (DE3) containning pET30a vector as comparison immuogene (negative immuogene) were emulsified with Freunds complete adjuvant respectively. 8-week-old clean class Balb/c female mice were immunized by hypodermic injection, 100μg per mouse. On the fourth and eighth week, the coequal dosage immunogenes emulsified with Freunds incomplete adjuvant were hypodermic injection. On the twelfth week, the immunogenes diluted by PBS were celiac injection, 150μg per mouse. After three days, the spleen cells of immunized mice were fused with myeloma cell line SP2/0 under PEG1500 and incubated in HAT culture liquid. After 10~15d, the cell liquids were determined by ELISA. ELISA plates were coated by the purified fusion proteins of bovine mature PrP and proteins of E. Coli JM09 (DE3) containning pET30a vector and the coated concentration was 25μg/mL.The antiserum(1:960) of mice immuned with positive immuogene and import mAb SAF70 (1:300) were positive comparison. The antiserum (1:960) of mice immuned with negative immuogene and SP2/0 cell liquids (1:1) were negative comparison. Goat anti Mouse IgG-HRP was the second antibody. The cells was thought of as positive clone as long as OD490nm value of the hybridoma cell liquids/negative comparison preponderated over three times. N64 hybridoma cell was limit diluted. After three subclone, N64 hybridoma cell was preserved in liquified nitrogen gas and cultivated continuously for three months to determined the stability of cell excretive antibody. To prepare ascites antibody, 1×10~6 N64 hybridoma cells were injected into pretreatmnt BALB/c mice by germfree liquid olefins. Adopting Protein A Agarose affinity chromatograghy, the ascites antibody could be purified. Using double-antibody layer ELISA, indirect ELISA and Western blot, Ig type and subtype, titer, relative affinity and specificity of the ascites antibody were determined and analyzed respectively. Finally, the epitope of the target proteins recognized by mAb was roughly identified through epitope mapping.Result: Monoclonal antibody (mAb) against PrPC mature protein of Qinchuan yellow cattle is obtained. The mAb belongs to IgG2a ,its titer is 1×10~5 and its relative affinity is 0.2μg/mL. Western blot demonstrates that the mAb has stronger specificity . Epitope analysis shows the mAb only reacts with carboxyl-terminal of recombinant PrP.更多还原