白喉毒素突变体CRM197的表达纯化及性质研究

发布时间：2018-04-06 23:02

本文选题：CRM197　切入点：蛋白载体　出处：《天津商业大学》2012年硕士论文

【摘要】：疫苗根据技术特点分为传统疫苗和新型疫苗。多糖结合疫苗则属于新型疫苗的一种。多糖结合疫苗是指采用化学方法将多糖共价结合在蛋白载体上所制备成的多糖-蛋白结合疫苗，用于提高细菌疫苗多糖抗原的免疫原性。国内研制的结合疫苗多采用类毒素，但类毒素作为载体可诱导产生全身及局部免疫反应，具有粘膜佐剂作用，但具有局限性。因为，用福尔马林或戊二醛脱毒的类毒素，会改变其自身的蛋白结构，可能破坏了重要的T细胞抗原位点，会影响其免疫原性。而且，体内存在的类毒素抗体可能影响结合疫苗的免疫应答。故现在研究的最多的蛋白载体是CRM197。 CRM197（cross reacting material197）是白喉毒素的一种突变体，它的第52位氨基酸由Gly突变为Glu，致使白喉毒素酶活性位点发生改变，从而不能对细胞产生毒性作用，但在抗原性和免疫原性上仍与天然的白喉毒素保持一致。因此本实验利用基因工程技术在大肠杆菌中对CRM197进行诱导表达，经纯化后，将重组蛋白CRM197作为载体，研究了它与肺炎链球菌荚膜多糖结合的可行性，并对其免疫原性进行了初步研究。首先，根据GenBank中白喉毒素的基因序列（登录号：CQ967258），进行密码子优化，加强分泌型信号肽，合成基因。同时设计引物，运用PCR技术扩增不含信号肽的CRM197基因。构建两组质粒pBAD-DEST49/CRM197和*pBAD-DEST49/CRM197。双酶切鉴定后，转入大肠杆菌中进行诱导表达。结果显示，分泌型菌株中的重组蛋白CRM197一部分存在于细胞间隙，，一部分以包涵体形式存在细胞质内。其次，对表达的蛋白分开纯化。分泌到细胞间隙的蛋白，通过增强细胞壁的通透性，让细胞间隙中的蛋白释放出来。对于包涵体形式的蛋白，破碎菌体离心后取沉淀。经过蛋白质变性复性等一系列步骤后，过DEAE阴离子柱，可使重组蛋白CRM197纯度达到95%以上。通过免疫印迹法验证重组CRM197的活性，结果显示重组蛋白CRM197具有良好的抗原性。最后，用溴化氰活化肺炎链球菌荚膜多糖，以ADH作为连接剂，以重组蛋白CRM197作为蛋白载体，在EDAC的作用下制备肺炎链球菌荚膜多糖-CRM197结合物。通过免疫双扩散试验证明结合物能分别与肺炎链球菌血清和白喉抗毒素产生沉淀线，说明结合物偶联成功，并具有良好的抗原性。本研究应用基因工程技术获得了能表达重组蛋白CRM917的工程菌菌株，并将纯化后的重组蛋白CRM197与肺炎链球菌荚膜多糖偶联，制成了肺炎链球菌荚膜多糖-CRM197结合物。为进一步以重组CRM197为载体蛋白，制备其它结合疫苗奠定基础。
[Abstract]:Vaccines are divided into traditional vaccines and new vaccines according to their technical characteristics.Polysaccharide-bound vaccine is a new type of vaccine.Polysaccharide bound vaccine is a kind of polysaccharide-protein binding vaccine which is prepared by chemical method. It is used to improve the immunogenicity of polysaccharide antigen of bacterial vaccine.Most of the conjugated vaccines in China use toxoid, but toxin as carrier can induce systemic and local immune response, which has mucosal adjuvant effect, but it has limitation.Because the toxin which is detoxified by formalin or glutaraldehyde can change its own protein structure, it may destroy the important T cell antigen site and affect its immunogenicity.Moreover, the presence of toxoid antibodies in the body may affect the immune response of the conjugated vaccine.So the most studied protein vector is CRM 197.CRM197(cross reacting material 197 is a mutant of diphtheria toxin. Its 52nd amino acid mutated from Gly to Glu. which caused diphtheria toxin activity site to change, so it could not produce toxic effect on cells.But antigenicity and immunogenicity remain consistent with natural diphtheria toxins.The recombinant protein CRM197 was used as a carrier to study the feasibility of the recombinant protein CRM197 binding to the capsule polysaccharide of Streptococcus pneumoniae.Its immunogenicity was studied preliminarily.Firstly, according to the gene sequence of diphtheria toxin in GenBank (accession number: CQ967258), the codon was optimized, the secretory signal peptide was enhanced, and the gene was synthesized.At the same time, primers were designed to amplify the CRM197 gene without signal peptide by PCR.Two sets of plasmids pBAD-DEST49/CRM197 and pBAD-DEST49 / CRM197were constructed.After double enzyme digestion, it was transferred into Escherichia coli for induction expression.The results showed that the recombinant protein CRM197 in the secretory strain was partly present in the intercellular space and in the cytoplasm in the form of inclusion body.Secondly, the expressed protein was purified separately.Proteins secreted into the intercellular space release the proteins from the intercellular space by enhancing the permeability of the cell wall.For proteins in the form of inclusion bodies, crushed bacteria were centrifuged and precipitated.After a series of steps such as protein denaturation and renaturation, the purity of recombinant protein CRM197 was over 95% after DEAE anion column.The activity of recombinant CRM197 was verified by Western blot. The results showed that the recombinant protein CRM197 had good antigenicity.Finally, the conjugate of Streptococcus pneumoniae capsule polysaccharide (CRM197) was prepared by using Cyanogen bromide as the activating agent of streptococcus pneumoniae capsule, ADH as the conjugate, and the recombinant protein CRM197 as the protein carrier under the action of EDAC.The double immunodiffusion test showed that the conjugate could produce precipitate line with Streptococcus pneumoniae serum and diphtheria antitoxin respectively, which indicated that the conjugate was successfully coupled and had good antigenicity.In this study, an engineering strain capable of expressing recombinant protein CRM917 was obtained by genetic engineering technique. The purified recombinant protein CRM197 was coupled with the capsular polysaccharide of Streptococcus pneumoniae to form the conjugate of Streptococcus pneumoniae capsule polysaccharide-CRM197.It lays a foundation for the preparation of other conjugated vaccines using recombinant CRM197 as carrier protein.
【学位授予单位】：天津商业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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