TRX单抗的制备及其在丙肝桥式双抗原夹心法中的初步应用

发布时间：2018-05-25 15:03

本文选题：TRX + 单克隆抗体　；参考：《郑州大学》2007年硕士论文

【摘要】： 丙型肝炎是感染丙型肝炎病毒(Hepatitis C virus，HCV)引起的传染性疾病。全球估计约1.7亿人感染HCV，中国感染人数约有3700万。部分HCV感染者将发展成为慢性肝炎、肝硬化、肝细胞癌。因此，HCV的早期诊断显得尤为重要。 HCV的实验室诊断方法主要有：抗-HCV检测、HCVAg检测、重组免疫印迹分析(RIBA)、HCV RNA检测等。其中，，抗HCV检测试剂应用最为广泛。目前主流的第三代抗HCV检测试剂均采用间接ELISA，而间接ELISA普遍存在假阳性和漏检的问题。与常规抗-HCV检测方法相比，双抗原夹心ELISA法可大大提高检测的特异性和敏感性。不但能检测到IgG抗体，而且还能够检测到早期出现的IgM抗体。然而双抗原夹心ELISA试剂迄今尚未研制成功。其原因在于HCV抗原经酶标记后，形成空间位阻，阻碍了抗体与抗原的结合。因此，直接标记抗原用于建立双抗原夹心ELISA不容易成功。硫氧还蛋白(thioredoxins，TRX)是分子量为12kDa的小分子蛋白质，可在大肠杆菌中高度表达并具有高度亲水性，能为丙肝抗原可溶性表达提供所必需的信号。据此制备出HCVAg-trx融合蛋白，若再以TRX作为桥蛋白，制备桥蛋白的单克隆抗体，用辣根过氧化物酶(HRP)标记。则由于桥蛋白及单抗的存在，标记的酶将不再阻碍抗原抗体的结合，能够较好地解决位阻的问题。目的本课题针对现有丙型肝炎诊断试剂的缺陷，通过表达、纯化TRX(桥蛋白)；制备TRX(桥蛋白)单克隆抗体，进行生物学鉴定。用辣根过氧化物酶(HRP)标记，为研制丙肝双抗原夹心ELISA试剂奠定基础。方法 1．TRX的表达、纯化将含有TRX基因的大肠杆菌经IPTG诱导表达4小时，收集菌体，冰浴下超声破菌，用亲和层析法纯化目的蛋白，12％SDS-PAGE鉴定。 2．杂交瘤细胞株的研制以纯化TRX为抗原免疫8周龄的雌性BALB／c小鼠，三次免疫后，取免疫鼠脾细胞与SP2／0骨髓瘤细胞以6:1的比例融合。在HAT选择培养基中选择培养，用间接ELISA法筛选阳性细胞，有限稀释法克隆和亚克隆，筛选出单克隆细胞株。 3．单克隆抗体的制备腹腔接种杂交瘤细胞，7-10天后采集腹水，并用正辛酸法以及离子层析纯化，10％SDS-PAGE鉴定。 4．杂交瘤细胞及单克隆抗体的鉴定用秋水仙素阻抑法使细胞阻滞于分裂中期，显微镜下观察，染色体计数，对杂交瘤细胞株进行染色体分析；采用间接ELISA测定单抗腹水效价、单抗相对亲和力；采用间接ELISA和双向免疫扩散法鉴定免疫球蛋白类型及亚类；以改良过碘酸钠法制备酶标抗体并作偶联物免疫活性的测定；以阻断抑制法作TRX单抗识别抗原表位的测定。 5．TRX单抗与丙肝融合抗原(HCVAg-trx)反应测定以间接ELISA法和Western blotting鉴定单克隆抗体与丙肝融合抗原(HCVAg-trx)的特异性反应。结果 1．TRX的表达、纯化结果12％SDS-PAGE分析表明目的蛋白得到了高表达，分离效果较好，纯度达到95％。 2．杂交瘤细胞株的研制获得2株能稳定分泌抗TRX单克隆抗体的杂交瘤细胞株，分别命名为2E8、5D6。 3．单克隆抗体的制备经紫外分光光度计测定计算，2E8、5D6单抗的浓度分别为1.55mg／mL、0.839 mg／mL。10％SDS-PAGE结果显示：纯化单抗样品在分子量约为150KD的位置出现有明显的电泳条带，为目的条带，纯度达到80％。 4．杂交瘤细胞及单克隆抗体的鉴定 (1)2E8、5D6的染色体平均条数分别为98、102，符合杂交瘤细胞染色体数约等于小鼠脾细胞和SP2／0骨髓瘤细胞染色体条数之和的理论值，由此证明所制备的细胞为杂交瘤细胞。 (2)两株单抗腹水的效价均为1:10~7；相对亲和力分析表明：5D6的相对亲和力高于2E8。 (3)两株单抗均为IgG类，IgG1亚类。 (4)2E8酶标抗体的免疫活性达到1:100×2~5，5D6酶标抗体达1:100×2~6。 (5)2E8和5D6两株单抗相互阻断，证明两者识别的抗原表位显著相关。 5．TRX单抗与重组丙肝抗原(HCVAg-trx)反应两株单抗均能与TRX及HCVAg-trx发生特异性结合，而不与无关蛋白IFN-γ产生交叉反应。结论 1．采用IPTG诱导表达，亲和层析法纯化，获得高纯度的重组TRX。 2．成功运用杂交瘤技术获得两株能稳定分泌抗TRX的杂交瘤细胞株。 3．成功对杂交瘤细胞株和纯化后的抗体进行了鉴定。 4．两株单抗均能够与丙肝融合抗原发生特异性结合反应。
[Abstract]:Hepatitis C is an infectious disease caused by hepatitis C virus ( HCV ) . Globally , about 170 million people have been infected with HCV , with a population of about 37 million . Some of the patients with HCV infection will develop into chronic hepatitis , cirrhosis and hepatocellular carcinoma . Therefore , early diagnosis of HCV is particularly important .

Anti - HCV detection , HCVAg detection , recombinant immune blotting analysis ( RIBA ) , HCV RNA detection , etc . The anti - HCV detection reagent is the most widely used .

Thioredoxins ( TRx ) is a small molecule protein with a molecular weight of 12 kDa , which can be highly expressed in E . coli and has high hydrophilicity . It can provide the necessary signal for the soluble expression of hepatitis C antigen .

Purpose

aiming at the defects of the prior hepatitis C diagnostic reagent , by expressing and purifying the trx ( bridge protein ) ;
Preparation of monoclonal antibody of trx ( bridge protein ) was carried out and biological identification was carried out . Using horseradish peroxidase ( HRP ) label , this paper lays a foundation for the development of HCV double antigen sandwich ELISA reagent .

method

1 . The expression of trx was purified and the E . coli containing trx gene was induced to express 4 hours by IPTG . The bacteria were collected , the ultrasonic bacteria were broken down under ice bath , the target protein was purified by affinity chromatography , and the 12 % SDS - PAGE was identified .

2 . The hybridoma cell line was prepared by immunizing eight - week old female BALB / c mice with purified trx . After three immunization , the spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells in a ratio of 6 : 1 . The positive cells were selected by indirect ELISA , the clones and subclones were screened by indirect ELISA , and the monoclonal cell lines were screened .

3 . The hybridoma cell was inoculated intraperitoneally with monoclonal antibody . The ascites was collected 7 - 10 days later and purified by means of n - octanol and ion chromatography . 10 % SDS - PAGE was used to identify the ascites .

4 . The hybridoma cell and the monoclonal antibody were used to identify the cell block in metaphase and metaphase , and the chromosome count was observed under the microscope and the chromosome analysis was carried out on the hybridoma cell line .
Indirect ELISA was used to determine the titer of monoclonal antibody and the relative affinity of monoclonal antibody .
Indirect ELISA and two - way immunodiffusion method were used to identify immunoglobulin type and subclass ;
preparing enzyme - labeled antibody by using modified sodium persulfate method and measuring the immunological activity of the conjugate ;
In this paper , the blocking suppression method was used to determine the epitope of the antigen epitope .

5 . The specific reaction of monoclonal antibody against hepatitis C fusion antigen ( HCVAg - trx ) was determined by indirect ELISA and Western blotting .

Results

1 . The results of SDS - PAGE analysis showed that the target protein was highly expressed , and the purity reached 95 % .

2 . The hybridoma cell lines secreting anti - trx monoclonal antibodies were obtained by the hybridoma cell line . The hybridoma cell lines were named 2e8 and 5D6 , respectively .

3 . The preparation of monoclonal antibody was determined by ultraviolet spectrophotometer . The concentration of the monoclonal antibody was 1.55 mg / mL and 0.839 mg / mL . 10 % SDS - PAGE showed that the purified monoclonal antibody had obvious electrophoretic bands at the position of about 150KD , and the purity reached 80 % .

4 . Identification of hybridoma cells and monoclonal antibodies

( 1 ) The average number of chromosome numbers of 2e8 and 5D6 was 98 , 102 , and the chromosome number of hybridoma cells was approximately equal to the theoretical value of chromosome number of mouse spleen cells and SP2 / 0 myeloma cells , thus demonstrating that the prepared cells were hybridoma cells .

( 2 ) the titer of the two monoclonal antibodies is 1 : 10 - 7 ;
The relative affinity analysis showed that the relative affinity of 5D6 was higher than that of 2E7 .

( 3 ) Both monoclonal antibodies were IgG , IgG1 subclass .

( 4 ) The activity of the enzyme labeled antibody of the enzyme was 1 : 100 脳 2 ~ 5 , the antibody of 5D6 enzyme was 1 : 100 脳 2 ~ 6 .

( 5 ) Two monoclonal antibodies were blocked from each other , which showed that the epitopes were significantly correlated with the epitope recognized by the two strains .

5 . Both monoclonal antibodies and recombinant hepatitis C antigen ( HCVAg - trx ) were able to bind specifically to trx and HCVAg - trx without cross - reaction with unrelated protein IFN - 纬 .

Conclusion

1 . IPTG - induced expression is adopted , affinity chromatography is adopted to purify , and high - purity recombinant trx is obtained .

2 . A hybridoma cell line secreting anti - trx stably was obtained by using hybridoma technique successfully .

3 . The hybridoma cell line and purified antibody were identified successfully .

4 . Both monoclonal antibodies can specifically bind to the hepatitis C fusion antigen .
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】