目的 应用生物信息学方法预测质粒介导的AmpC β-内酰胺酶(pAmpCs)共有抗原表位,并实现pAmpCs共有抗原表位-Trx融合蛋白的原核表达、纯化和鉴定。 方法 运用多序列比较工具找出各型质粒介导的AmpC β-内酰胺酶的共有相对保守区域,并利用分子生物学软件Biosun预测各亚型pAmpCs的可及性、抗原性、抗原表位、柔性、亲水性等参数,通过综合分析后找出其共有的抗原表位区域pAmpCs1。根据大肠杆菌的密码子偏好性,把氨基酸序列pAmpCs1转变为核苷酸序列pampcs1,采用重叠延伸PCR合成pampcs1片断,并构建原核表达载体pET32a(+)/pampcs1。通过测序验证,对含有该重组质粒的大肠杆菌BL21(DE3)进行诱导表达,表达产物经SDS-PAGE电泳分析后,用Ni-NTA亲和层析法纯化pAmpCs1-Trx融合蛋白,利用western blotting方法鉴定融合蛋白的抗原性。 结果 根据预测结果找到了一段各型pAmpCs共有的抗原表位区域pAmpCs1,成功构建了原核表达载体pET32a(+)/pampcs1,经IPTG诱导后得到了分子量约为23KD的融合蛋白,经Ni-NTA亲和层析柱纯化后,通过western blotting鉴定该蛋白能被抗AmpC β-lactamases多抗识别。 结论 成功构建了pAmpCs1-Trx融合蛋白原核表达载体,并获得了高纯度的pAmpCs1-Trx融合蛋白,初步证明它的抗原性,为制备特异性抗体及建立快速检测方法打下了良好的基础。
Objective: Using the method of bioinformatics to predict the mutual epitope of Plasmid-mediated AmpC β-lactamases (pAmpCs) and to realize prokaryotic expression, purifica- tion and identification of AmpCs-Trx fusion protein. Method: Using the multi-sequence compa- rison tool to find various types of pAmpCs's relative conservative region, and using molecular biology software Biosun to predict the parameters of the accessibility, the antigenicity, the epitope, the flexibility and the hopp-woods, etc . Through comprehensive analyzing, find out the area of mutual epitope pAmpCs1. Based on the preference of the E. coli codon, obtain the nucleotide sequence pampcs1. Then the pampcs1 fragment was synthesized using overlapping PCR and the expression vector which is called pET32a (+) / pampcs1 was constructed. Adopting sequence verified, to make induced expression for the E.coli BL21(DE3) which contains the recombinnant plasmid. After the expressed product was analyzed by SDS-PAGE, to purify pAmpCs1-Trx fusion protein by using Ni-NTA affinity chromatography. And using the method of Western blotting to identify the antigenicity of pAmpCs1. Result: According to the result of prediction, the area of mutual epitope have been found out, and successfully established the recombinant expression vector pET32a(+)/pampcs1. BL21(DE3) strain was induced by IPTG, a specific expression band with a relative molecular mass 23×103 was detected by SDS-PAGE,and the purified fusion protein can be detected by multiclonal antibody against AmpC β-lactamases. Conclusion: Having been successfully established AmpCs1-Trx fusion protein prokaryotic expression vector and having been obtained the highly purified AmpCs1-Trx fusion protein. Initially prove the antigenicity of the AmpCs1-Trx fusion protein , settle a good foundation for preparing specific antibodies against pAmpCs1 and detecting pAmpCs.
孔路科,郭建巍,马骢,杨林西,魏杰,吴素香. 质粒介导的AmpC β-内酰胺酶共有抗原表位的预测及原核表达[J]. 中国生物工程杂志, 2008, 28(9): 32-38.
. Prediction and Prokaryotic Expression of the Mutual Epitope from all Plasmid-mediatedAmpC β-lactamases. China Biotechnology, 2008, 28(9): 32-38.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2008/V28/I9/32
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