论文部分内容阅读
目的:构建EGFRvIII与HBcAg重组基因的原核表达质粒,进行原核表达、纯化,观察表达蛋白的免疫原性和抗原性。方法:通过双酶切从pGEM-TEasy/PEP-3载体获得编码EGFRvIII突变区的cDNA片段,插入去除了主要免疫优势区的重组质粒pGEMEX/HBcAg中,获得重组质粒pGEMEX/c-PEP-3-c,将重组基因亚克隆于原核表达载体pET28a(+)中,通过IPTG诱导蛋白表达,利用Ni2+-NTA亲和层析法对融合蛋白进行纯化;用融合蛋白常规免疫BALB/c小鼠,用间接ELISA检测小鼠血清中抗体的滴度。结果:经酶切鉴定、序列测定证实融合基因已插入pET28a(+)载体中,融合基因序列与设计序列完全一致。原核表达后表达量占沉淀全菌蛋白的56%,纯化产物占总蛋白的92%,浓度约8g/L。BALB/c小鼠经4次免疫后,其血清中中和抗体的滴度可达1∶16000,第6次免疫后血清中中和抗体的滴度达到1∶2.56×105,抗PEP-3抗体滴度达到1∶1.28×105,抗HBcAg抗体滴度小于1∶4×103。结论:融合基因PEP-3-HBcAg在大肠杆菌中可获得高效表达,表达的融合蛋白可以诱导小鼠产生高滴度和高特异性中和抗体,从而为高表达EGFRvIII恶性肿瘤基因工程疫苗的研究奠定基础。
OBJECTIVE: To construct prokaryotic expression plasmids of recombinant EGFRvIII and HBcAg, prokaryotic and purify them and observe the immunogenicity and antigenicity of the expressed proteins. Methods: The cDNA fragment encoding the EGFRvIII mutation was obtained by double digestion from the pGEM-TEasy / PEP-3 vector and inserted into the recombinant plasmid pGEMEX / HBcAg with the major immunodominant region removed to obtain the recombinant plasmid pGEMEX / c-PEP- The recombinant protein was subcloned into the prokaryotic expression vector pET28a (+), and the protein was induced by IPTG. The fusion protein was purified by Ni2 + -NTA affinity chromatography. BALB / c mice were immunized with the fusion protein routinely Indirect ELISA was used to detect the titer of antibody in mouse serum. Results: The results of restriction enzyme digestion showed that the fusion gene was inserted into pET28a (+) vector and the fusion gene sequence was exactly the same as the designed sequence. Prokaryotic expression accounted for 56% of the total precipitated protein, the purified product accounted for 92% of the total protein, the concentration of about 8g / L. BALB / c mice after four immunizations, the serum titer of neutralizing antibody up to 1:16000, after the sixth immunization, serum neutralizing antibody titer reached 1: 2.56 × 105, anti-PEP-3 Antibody titer reached 1: 1.28 × 105, anti-HBcAg antibody titer was less than 1: 4 × 103. CONCLUSION: The fusion gene PEP-3-HBcAg is highly expressed in E. coli, and the expressed fusion protein can induce high titers and high specific neutralizing antibodies in mice, so as to be a genetically engineered vaccine highly expressing EGFRvIII malignant tumor Lay the foundation.