减毒沙门氏菌运载重组G-LTB狂犬疫苗口服免疫小鼠的研究

发布时间：2018-04-26 18:36

本文选题：狂犬病 + 沙门氏菌　；参考：《吉林大学》2011年硕士论文

【摘要】：狂犬病毒G基因能够表达分子量为67 kd的由524个氨基酸组成的糖蛋白,糖蛋白是唯一诱导机体产生中和抗体和保护性免疫应答的主要结构蛋白,其免疫作用与全病毒疫苗相当。膜外区决定狂犬病毒糖蛋白的抗原性、组织亲嗜性及毒力。它既是有效的保护性抗原,也能诱导产生中和抗体和刺激细胞免疫。热不稳定大肠杆菌肠毒素(LT)由一个A亚单位和一个经5个非共价键连接形成的五聚体B亚单位构成。研究表明,LTB是一种良好的黏膜免疫佐剂,具有很强的免疫调节作用,当与其他抗原共刺激机体后,LTB可促使机体产生特异的针对共刺激抗原的抗体而表现出佐剂活性。近年来,诸多研究已证实鼠伤寒沙门氏菌可作为细菌,病毒,寄生虫和肿瘤疫苗抗原的理想载体,其所携带外源基因能够高效表达并能全面激发机体的体液与细胞免疫。因此,以鼠伤寒沙门氏菌为基础的载体在疫苗研究领域备受国内外众多学者的关注。本研究利用生物信息学软件对狂犬病毒糖蛋白基因以及热不稳定肠毒素B亚单位基因进行生物信息学分析。从GenBank调取G和LTB基因序列并根据Oligo-6设计2对引物,以pMD-G和pMD-LTB为模板,通过PCR的方法分别扩增G-linker-片段,-linker-LTB片段,然后又以其PCR产物为模板,采用重叠延伸PCR的方法扩增出G-LTB的融合基因,然后将该融合基因经T-A连接反应连接到pMD18-T克隆载体上。质粒pMD-G-LTB和pVAX1、pVAX1-ori载体经BamHⅠ和XhoⅠ双酶切后,将所获得的G-LTB融合基因和载体经T4DNA连接酶连接。经PCR、酶切鉴定结果正确,测序鉴定表明,外源基因核酸序列没有发生变异,和Genbank上公布的基因序列一致。将获得的重组质粒pVAX1-G-LTB和pVAX1-ori-G-LTB转化到大肠杆菌JM109中,质粒提取,转染BHK-21细胞中。培养48h后,进行间接免疫荧光检测分析,结果表明,真核表达质粒在BHK-21中表达了狂犬病病毒G-LTB融合蛋白。Western blot检测可发现一条68.8kd左右的目的带,证明了本实验成功构建了真核表达质粒pVAX1-G-LTB和pVAX 1-ori-G-LTB载体。为了进一步检测G-LTB融合基因的免疫原性,将pVAX1, pVAX1-G, pVAX-1-G-LTB, pVAX1-ori-G-LTB电转化减毒沙门氏菌突变株phoP/phoQ中,构建的重组株,均以1010 CFU的剂量口服免疫6-8周龄的雌性BALB/C小鼠。间隔10天免疫一次,共免疫两次。狂犬病毒IgG抗体检测实验表明phoP/phoQ(pVAX1-G-LTB)和p-hoP/phoQ(pVAX1-ori-G-LTB)免疫组的抗体水平明显高于控制组。中和抗体检测表明抗体水平大于0.5IU/mL,而对照组和PBS组均低于0.5IU/mL。动物保护性试验表明phoP/phoQ(pVAX1-G-LTB)和phoP/phoQ(pVAX1-ori-G-LTB)组分别达到了50%、60%的保护率。IL-2水平检测结果同上,统计学分析差异明显。本研究结果表明,以减毒沙门氏菌为载体运载G-LTB基因的口服疫苗能诱导小鼠产生抗狂犬病毒的体液和细胞免疫应答。同肌肉注射或基因枪等免疫方式相比口服疫苗省时、方便、价廉、易于群体免疫,是研制低成本、实用化口服DNA疫苗的一条新途径。因此本研究将为进一步研发出基于粘膜免疫途径的新型狂犬口服疫苗奠定了基础。
[Abstract]:The rabies virus G gene is capable of expressing a glycoprotein composed of 524 amino acids having a molecular weight of 67 kd . The glycoprotein is the only major structural protein that induces the body to generate neutralizing antibody and protective immune response . Its immune function is comparable to that of the whole virus vaccine . The outer region of the membrane determines the antigenicity , tissue tropism and virulence of the rabies virus glycoprotein . It is both an effective protective antigen and can induce neutralizing antibody and stimulate cell immunity .

Heat - labile Enterotoxin ( LT ) is composed of a subunit A and a pentamer B subunit formed from five non - covalent bonds . The study shows that LTB is a good mucosal immune adjuvant and has a strong immunomodulatory effect . After co - stimulating the organism with other antigens , LTB can induce specific antibody against costimulatory antigen to show adjuvant activity .

In recent years , many researches have proved that Salmonella typhimurium can be used as an ideal carrier for bacteria , viruses , parasites and tumor vaccine antigens , which carry foreign genes efficiently and completely stimulate body fluid and cellular immunity . Therefore , the carrier based on Salmonella typhimurium has attracted much attention from many scholars in the field of vaccine research .

The fusion gene of G - LTB was amplified by PCR . The fusion gene of G - LTB was amplified by PCR .

In order to further detect the immunogenicity of the G - LTB fusion gene , the recombinant strains were immunized twice with a dose of 1010 CFU for 6 - 8 weeks old female BALB / C mice .

Compared with intramuscular injection or gene gun immunization , it is convenient , cheap and easy to group immunity . It is a new way to develop low - cost and practical oral DNA vaccine .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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