弓形虫多表位基因的疫苗研究及其重组抗原在免疫诊断中的应用

发布时间：2018-04-13 06:35

本文选题：弓形虫 + 多表位基因　；参考：《第一军医大学》2007年博士论文

【摘要】： 第一部分弓形虫多表位疫苗研究 1．疫苗的制备构建弓形虫多表位基因在真核表达系统中的表达质粒pCDNA3-MAG。以试剂盒大量纯化质粒pcDNA3-MAG、pcDNA3-SAG1和pcDNA3，用于制备核酸疫苗。以脂质体作为免疫佐剂，增强疫苗的免疫效果。诱导并纯化弓形虫MAG的原核表达重组抗原(rMAG1)、SAG1的原核表达重组抗原(rSAG1)及载体质粒pET32a表达的硫氧还蛋白(Trx)，作为DNA疫苗的加强免疫疫苗。 2．动物免疫以BALB／C小鼠为免疫对象，按照疫苗的构成和免疫方式不同，将实验动物分为DNA疫苗免疫系列和“基础-加强”免疫系列，前者均以MAG的DNA疫苗进行免疫，后者先以MAG的DNA疫苗免疫，再以重组抗原rMAG进行增强免疫。每系列均设SAG1免疫组、载体对照组和空白对照组。 3．免疫应答及免疫保护性小鼠免疫前及末次免疫后第1周、第3周、第5周，分别采集血清，以ELISA试剂盒检测各组小鼠的抗弓形虫IgG抗体、IFN—γ和IL—4含量，末次免疫后6w，以RH株弓形虫速殖子感染小鼠(200个／只)，统计各组小鼠的存活时间和存活率。综合各项检测指标进行统计分析后，结果如下： DNA疫苗实验结果显示，同免疫前、载体对照组和空白对照组相比，MAG的DNA疫苗刺激小鼠产生了特异性抗弓形虫IgG抗体和高水平IFN—γ，，而与SAG1组比较，无显著性差异；在对抗致死剂量弓形虫的感染上，同SAG1免疫组、载体对照组和空白对照组相比，MAG组小鼠不仅延长了存活期，还有1只存活90天以上(存活率为8.3％)。 DNA疫苗初免、重组抗原疫苗加强免疫的实验结果显示，同免疫前、载体对照组和空白对照组相比，MAG组小鼠产生了特异性抗弓形虫IgG抗体和高水平IFN—γ，与SAG1组比较，IFN—γ含量无显著性差异，但IgG抗体水平有显著差异，MAG组高于SAG1组；在对抗致死剂量弓形虫的感染上，同载体对照组和空白对照组相比，MAG组小鼠不仅显著延长了存活时间，还有4只存活90天以上(存活率为33.3％)，同SAG1免疫组相比，小鼠存活时间无显著性差异，但存活率高于SAG1组的16.7％。就MAG疫苗的两种不同免疫方式——DNA疫苗单独免疫和DNA疫苗初免、重组抗原疫苗加强免疫——进行比较，二者激发的体液、细胞免疫存在非常显著性差异，后者显著高于前者；在对抗致死剂量弓形虫的感染上，两组小鼠的存活时间具有非常显著性差异，后者显著高于前者；两组均有小鼠存活存活90天以上，前者为1只，存活率为8.3％，后者存活4只，存活率为33.3％。结果显示先以DNA疫苗初免、再以重组抗原疫苗加强免疫，较单纯DNA免疫，获得了更好的免疫保护效果。实验中，以弓形虫强保护性抗原SAG1(P30)作为多表位疫苗的平行对照，结果表明多表位疫苗的免疫效果在整体上优于SAG1基因疫苗，尽管后者也获得了较好的免疫保护性(如以其基因的DNA疫苗初免、以其重组抗原疫苗加强，使免疫的小鼠获得了16.7％的存活率)。为了验证弓形虫多表位疫苗在免疫小鼠后各表位的表达和呈递，分别制备了三种重组抗原rSAG1、rGRA2、rROP2，以Western-blot检测小鼠免疫血清和上述重组抗原的特异性结合反应。结果显示，以两种方式免疫后的小鼠血清，均可被上述三种重组抗原所特异识别，表明多表位基因所包含的表位在小鼠体内得到了有效的表达和呈递，并激发了特异的体液免疫反应。本实验中，以弓形虫RH株感染小鼠，空白对照组小鼠在7天内全部死亡。而以MAG的基因疫苗免疫小鼠，小鼠的存活时间显著延长，且有8.3％的小鼠存活90天以上；以DNA疫苗初免、重组抗原疫苗加强免疫小鼠，33.3％的的小鼠存活90天以上。以上结果表明该疫苗具有良好的免疫保护性，验证了以多表位的方法制备弓形虫病疫苗、以复合免疫的方式增强免疫效果的可行性。第二部分重组多表位抗原在免疫诊断中的初步应用 1．弓形虫感染小鼠血清的制备及鉴定以B36株弓形虫感染小鼠，采集血清，以ELISA和Western—blot检测抗弓形虫IgG抗体，直接镜检脑内包囊或腹腔速殖子，共获得17只小鼠的感染血清，效价最高可达1：51200。 2．弓形虫多表位重组抗原的可溶性表达、纯化及鉴定大量诱导表达可溶性的弓形虫多表位重组抗原(rMAG)，以Ni-NTA agarose和割胶电渗对其进行两步法纯化，最后得到了纯度达95.86％的rMAG。免疫印迹实验证明，弓形虫多表位重组可溶性抗原不仅能够被弓形虫强毒株RH感染的兔血清及抗弓形虫速殖子主要表面抗原SAG1(P30)的单抗所识别，而且能被成囊株弓形虫B36株慢性感染的小鼠血清所识别，提示该抗原既含有弓形虫速殖子抗原表位，也含有缓殖子包囊抗原表位。 3．弓形虫多表位重组抗原在检测弓形虫感染中的应用以弓形虫感染血清为一抗，筛选重组弓形虫多表位抗原的最佳包被浓度为3μg／ml，用于制备ELISA检测试剂盒。检测小鼠血清141份，其中阳性小鼠感染血清为117份、正常小鼠血清24份，结果显示，该试剂盒敏感性为88.88％(104／117×100％)，特异性为91.67％(22／24×100％)，一致性为89.36％((104+22)／141×100％)。检测兔血清24份，其中急性感染弓形虫的兔血清18份，正常兔血清6份，阳性血清检出率为94.4％，总一致性为91.5％((17+5)／24×100％)。以上结果显示，弓形虫多表位抗原试剂盒既可以检测弓形虫急性感染血清，又可以检测弓形虫慢性感染血清，初步证实了多表位抗原用于弓形虫感染诊断中的优势，为试剂盒的下一步应用奠定了基础。
[Abstract]:The first part of the multi epitope vaccine of Toxoplasma gondii
Preparation of 1. vaccine
To construct the expression plasmid pCDNA3-MAG. of Toxoplasma gondii multi epitope gene in eukaryotic expression system, we purified plasmid pcDNA3-MAG, pcDNA3-SAG1 and pcDNA3 with kit to prepare nucleic acid vaccine. Liposomes were used as adjuvants to enhance the immune effect of vaccines.
The prokaryotic expression recombinant antigen (rMAG1) of Toxoplasma gondii MAG was induced and purified, the prokaryotic expression recombinant antigen (rSAG1) of SAG1 and the thioredoxin (Trx) expressed by plasmid pET32a were used as an enhanced vaccine for DNA vaccine.
2. animal immunity
In BALB / C mice as immune object, according to the different composition and immunity of the vaccine, the experimental animal is divided into series of DNA vaccine and immune based boost immunity to the former series, MAG DNA vaccine, the first to DNA vaccine MAG, then recombinant antigen rMAG to enhance immunity. Each series are equipped with SAG1 immune group, vector control group and blank control group.
3. immune response and immunological protection
First weeks, mice before and third weeks after the final immunization, fifth weeks, serum were collected with anti Toxoplasma IgG antibody detection kit ELISA mice, IFN - ray and IL - 4 content, after the last immunization of 6W mice infected with Toxoplasma gondii RH strain tachyzoites (200 / only), the mice survival time and survival rate. The comprehensive indexes for statistical analysis, the results are as follows:
The experimental results show that with DNA vaccine, immune, vector control group compared with blank control group, MAG DNA vaccine stimulated mice produced specific anti Toxoplasma IgG antibody and the high level of IFN gamma, and compared with the SAG1 group, no significant difference; in the fight against a lethal dose of Toxoplasma infection on the same. SAG1 immune group, vector control group compared with blank control group, MAG group of mice can prolong the survival period, and 1 of them survived more than 90 days (the survival rate was 8.3%).
DNA vaccines, recombinant antigen vaccine immunization experiments showed that the same before immunization, vector control group compared with blank control group, MAG mice produced specific anti Toxoplasma IgG antibody and the high level of IFN gamma, compared with group SAG1, IFN had no significant difference but significant gamma content. The difference of IgG antibody level of MAG group was higher than that of SAG1 group; in the fight against a lethal dose of Toxoplasma infection on the same carrier control group compared with blank control group, MAG group were not only significantly prolonged survival time, and 4 of them survived more than 90 days (survival rate 33.3%), compared with the SAG1 group, no significant difference differences in survival time of mice, but the survival rate is higher than that of SAG1 group 16.7%.
The MAG vaccine of two different immune method -- DNA vaccine alone and immune DNA vaccines, recombinant antigen vaccine -- comparison of two excitation fluids, there exists significant difference in cellular immunity, which was significantly higher than the former; in the fight against a lethal dose of arch insect infection, two mice survival time has a very significant difference, which was significantly higher than the former; all two survival mice survived more than 90 days, the former is 1, the survival rate was 8.3%, the latter 4 survived, the survival rate was 33.3%. showed by DNA vaccines, recombinant vaccines to strengthen immunity, compared with DNA, immune, get the immune protection effect better.
In the experiment, with the strong protective antigen SAG1 of Toxoplasma gondii (P30) as multi epitope vaccine showed that parallel controlled, multi epitope vaccine immune effect is superior to the SAG1 gene vaccine results, although the latter also received a good immune protection (such as the gene DNA vaccines, with its recombinant strengthen the immune antigen vaccine, the mice survival rate of 16.7%) obtained.
In order to verify the multi epitope vaccine of Toxoplasma gondii in mice after immunization with the epitope expression and presentation, respectively, prepared three kinds of recombinant antigen rSAG1, rGRA2, rROP2, Western-blot were detected with specific immune serum and the recombinant antigen binding reaction. The results showed that in two ways after immunization of mice serum. Can be the three recombinant antigen specific recognition, show that the table contains the epitope gene in mice by expression and presentation effectively, and to stimulate specific humoral immune response.
In this experiment, the RH strain of Toxoplasma gondii infected mice, the mice in blank group all died within 7 days. The DNA vaccine MAG in mice, the survival time of mice was significantly prolonged, and 8.3% of the mice survived more than 90 days; to DNA vaccines, recombinant vaccines to enhance immunity in mice, 33.3% the mice survived more than 90 days. The above results showed that the vaccine has good immune protection, to verify the multi epitope preparation method of toxoplasmosis vaccine, immune to the feasibility of composite immune effect.
The preliminary application of the second part of recombinant multiple epitope antigen in the immunodiagnosis
Preparation and identification of sera from mice infected with 1. Toxoplasma gondii
Toxoplasma gondii B36 infected mice were collected. Serum and ELISA and Western blot were used to detect IgG antibodies against Toxoplasma gondii. The cysts or cysts in the brain were directly examined. The serum of 17 mice was obtained. The highest titer was 1:51200..
Soluble expression, purification and identification of the multi epitope antigen of 2. Toxoplasma gondii
Expression of Toxoplasma gondii soluble recombinant multi epitope antigen (rMAG), the two step purification with Ni-NTA agarose and tapping electroosmosis, finally obtained the proof of rMAG. Western blot. The purity of 95.86% of Toxoplasma gondii recombinant multi epitope antigen of Toxoplasma gondii can be virulent strain RH infected rabbit serum and the main the surface antigen of Toxoplasma gondii tachyzoites (SAG1 P30) of the mAbs, and serum of mice can be encapsulated strains of Toxoplasma gondii B36 strain of chronic infection identified, this antigen containing Toxoplasma tachyzoite antigen epitope, also contain bradyzoite cysts epitope.
The application of multi epitope antigen of Toxoplasma 3. in detection of Toxoplasma gondii infection
The optimal concentration of recombinant Toxoplasma antigen was 3 g / ml, which was used for the preparation of ELISA detection kit.
A total of 141 serum samples were detected, including 117 sera from positive mice and 24 sera from normal mice. The results showed that the sensitivity of the kit was 88.88% (104 / 117 x 100%), the specificity was 91.67% (22 / 24 x 100%), and the consistency was 100% (104+22) / 100% *
Serum samples from 24 rabbits were collected, including 18 rabbits infected with Toxoplasma gondii and 6 sera from normal rabbits. The positive rate of serum was 94.4%, and the total consistency was 91.5% ((17+5) / 24 x 100%).
The above results showed that Toxoplasma gondii multiepitope antigen kit can detect acute toxoplasma infection in serum, and detection of chronic infection of Toxoplasma gondii serum, confirmed that multiple epitope antigen for diagnosis of Toxoplasma gondii infection in the advantage, laid the foundation for the next step in the application kit.

【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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