本文選題：CRM197　切入點：蛋白載體　出處：《天津商業(yè)大學(xué)》2012年碩士論文

【摘要】：疫苗根據(jù)技術(shù)特點分為傳統(tǒng)疫苗和新型疫苗。多糖結(jié)合疫苗則屬于新型疫苗的一種。多糖結(jié)合疫苗是指采用化學(xué)方法將多糖共價結(jié)合在蛋白載體上所制備成的多糖-蛋白結(jié)合疫苗，用于提高細菌疫苗多糖抗原的免疫原性。國內(nèi)研制的結(jié)合疫苗多采用類毒素，但類毒素作為載體可誘導(dǎo)產(chǎn)生全身及局部免疫反應(yīng)，具有粘膜佐劑作用，但具有局限性。因為，用福爾馬林或戊二醛脫毒的類毒素，會改變其自身的蛋白結(jié)構(gòu)，可能破壞了重要的T細胞抗原位點，會影響其免疫原性。而且，體內(nèi)存在的類毒素抗體可能影響結(jié)合疫苗的免疫應(yīng)答。故現(xiàn)在研究的最多的蛋白載體是CRM197。 CRM197（cross reacting material197）是白喉毒素的一種突變體，它的第52位氨基酸由Gly突變?yōu)镚lu，致使白喉毒素酶活性位點發(fā)生改變，從而不能對細胞產(chǎn)生毒性作用，但在抗原性和免疫原性上仍與天然的白喉毒素保持一致。因此本實驗利用基因工程技術(shù)在大腸桿菌中對CRM197進行誘導(dǎo)表達，經(jīng)純化后，將重組蛋白CRM197作為載體，研究了它與肺炎鏈球菌莢膜多糖結(jié)合的可行性，并對其免疫原性進行了初步研究。 首先，根據(jù)GenBank中白喉毒素的基因序列（登錄號：CQ967258），進行密碼子優(yōu)化，加強分泌型信號肽，合成基因。同時設(shè)計引物，運用PCR技術(shù)擴增不含信號肽的CRM197基因。構(gòu)建兩組質(zhì)粒pBAD-DEST49/CRM197和*pBAD-DEST49/CRM197。雙酶切鑒定后，轉(zhuǎn)入大腸桿菌中進行誘導(dǎo)表達。結(jié)果顯示，分泌型菌株中的重組蛋白CRM197一部分存在于細胞間隙，，一部分以包涵體形式存在細胞質(zhì)內(nèi)。 其次，對表達的蛋白分開純化。分泌到細胞間隙的蛋白，通過增強細胞壁的通透性，讓細胞間隙中的蛋白釋放出來。對于包涵體形式的蛋白，破碎菌體離心后取沉淀。經(jīng)過蛋白質(zhì)變性復(fù)性等一系列步驟后，過DEAE陰離子柱，可使重組蛋白CRM197純度達到95%以上。通過免疫印跡法驗證重組CRM197的活性，結(jié)果顯示重組蛋白CRM197具有良好的抗原性。 最后，用溴化氰活化肺炎鏈球菌莢膜多糖，以ADH作為連接劑，以重組蛋白CRM197作為蛋白載體，在EDAC的作用下制備肺炎鏈球菌莢膜多糖-CRM197結(jié)合物。通過免疫雙擴散試驗證明結(jié)合物能分別與肺炎鏈球菌血清和白喉抗毒素產(chǎn)生沉淀線，說明結(jié)合物偶聯(lián)成功，并具有良好的抗原性。 本研究應(yīng)用基因工程技術(shù)獲得了能表達重組蛋白CRM917的工程菌菌株，并將純化后的重組蛋白CRM197與肺炎鏈球菌莢膜多糖偶聯(lián)，制成了肺炎鏈球菌莢膜多糖-CRM197結(jié)合物。為進一步以重組CRM197為載體蛋白，制備其它結(jié)合疫苗奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津商業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

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