本文選題：弓形蟲(chóng) + 多表位基因�。� 參考：《第一軍醫(yī)大學(xué)》2007年博士論文

【摘要】： 第一部分弓形蟲(chóng)多表位疫苗研究 1．疫苗的制備 構(gòu)建弓形蟲(chóng)多表位基因在真核表達(dá)系統(tǒng)中的表達(dá)質(zhì)粒pCDNA3-MAG。以試劑盒大量純化質(zhì)粒pcDNA3-MAG、pcDNA3-SAG1和pcDNA3，用于制備核酸疫苗。以脂質(zhì)體作為免疫佐劑，增強(qiáng)疫苗的免疫效果。 誘導(dǎo)并純化弓形蟲(chóng)MAG的原核表達(dá)重組抗原(rMAG1)、SAG1的原核表達(dá)重組抗原(rSAG1)及載體質(zhì)粒pET32a表達(dá)的硫氧還蛋白(Trx)，作為DNA疫苗的加強(qiáng)免疫疫苗。 2．動(dòng)物免疫 以BALB／C小鼠為免疫對(duì)象，按照疫苗的構(gòu)成和免疫方式不同，將實(shí)驗(yàn)動(dòng)物分為DNA疫苗免疫系列和“基礎(chǔ)-加強(qiáng)”免疫系列，前者均以MAG的DNA疫苗進(jìn)行免疫，后者先以MAG的DNA疫苗免疫，再以重組抗原rMAG進(jìn)行增強(qiáng)免疫。每系列均設(shè)SAG1免疫組、載體對(duì)照組和空白對(duì)照組。 3．免疫應(yīng)答及免疫保護(hù)性 小鼠免疫前及末次免疫后第1周、第3周、第5周，分別采集血清，以ELISA試劑盒檢測(cè)各組小鼠的抗弓形蟲(chóng)IgG抗體、IFN—γ和IL—4含量，末次免疫后6w，以RH株弓形蟲(chóng)速殖子感染小鼠(200個(gè)／只)，統(tǒng)計(jì)各組小鼠的存活時(shí)間和存活率。綜合各項(xiàng)檢測(cè)指標(biāo)進(jìn)行統(tǒng)計(jì)分析后，結(jié)果如下： DNA疫苗實(shí)驗(yàn)結(jié)果顯示，同免疫前、載體對(duì)照組和空白對(duì)照組相比，MAG的DNA疫苗刺激小鼠產(chǎn)生了特異性抗弓形蟲(chóng)IgG抗體和高水平IFN—γ，，而與SAG1組比較，無(wú)顯著性差異；在對(duì)抗致死劑量弓形蟲(chóng)的感染上，同SAG1免疫組、載體對(duì)照組和空白對(duì)照組相比，MAG組小鼠不僅延長(zhǎng)了存活期，還有1只存活90天以上(存活率為8.3％)。 DNA疫苗初免、重組抗原疫苗加強(qiáng)免疫的實(shí)驗(yàn)結(jié)果顯示，同免疫前、載體對(duì)照組和空白對(duì)照組相比，MAG組小鼠產(chǎn)生了特異性抗弓形蟲(chóng)IgG抗體和高水平IFN—γ，與SAG1組比較，IFN—γ含量無(wú)顯著性差異，但I(xiàn)gG抗體水平有顯著差異，MAG組高于SAG1組；在對(duì)抗致死劑量弓形蟲(chóng)的感染上，同載體對(duì)照組和空白對(duì)照組相比，MAG組小鼠不僅顯著延長(zhǎng)了存活時(shí)間，還有4只存活90天以上(存活率為33.3％)，同SAG1免疫組相比，小鼠存活時(shí)間無(wú)顯著性差異，但存活率高于SAG1組的16.7％。 就MAG疫苗的兩種不同免疫方式——DNA疫苗單獨(dú)免疫和DNA疫苗初免、重組抗原疫苗加強(qiáng)免疫——進(jìn)行比較，二者激發(fā)的體液、細(xì)胞免疫存在非常顯著性差異，后者顯著高于前者；在對(duì)抗致死劑量弓形蟲(chóng)的感染上，兩組小鼠的存活時(shí)間具有非常顯著性差異，后者顯著高于前者；兩組均有小鼠存活存活90天以上，前者為1只，存活率為8.3％，后者存活4只，存活率為33.3％。結(jié)果顯示先以DNA疫苗初免、再以重組抗原疫苗加強(qiáng)免疫，較單純DNA免疫，獲得了更好的免疫保護(hù)效果。 實(shí)驗(yàn)中，以弓形蟲(chóng)強(qiáng)保護(hù)性抗原SAG1(P30)作為多表位疫苗的平行對(duì)照，結(jié)果表明多表位疫苗的免疫效果在整體上優(yōu)于SAG1基因疫苗，盡管后者也獲得了較好的免疫保護(hù)性(如以其基因的DNA疫苗初免、以其重組抗原疫苗加強(qiáng)，使免疫的小鼠獲得了16.7％的存活率)。 為了驗(yàn)證弓形蟲(chóng)多表位疫苗在免疫小鼠后各表位的表達(dá)和呈遞，分別制備了三種重組抗原rSAG1、rGRA2、rROP2，以Western-blot檢測(cè)小鼠免疫血清和上述重組抗原的特異性結(jié)合反應(yīng)。結(jié)果顯示，以兩種方式免疫后的小鼠血清，均可被上述三種重組抗原所特異識(shí)別，表明多表位基因所包含的表位在小鼠體內(nèi)得到了有效的表達(dá)和呈遞，并激發(fā)了特異的體液免疫反應(yīng)。 本實(shí)驗(yàn)中，以弓形蟲(chóng)RH株感染小鼠，空白對(duì)照組小鼠在7天內(nèi)全部死亡。而以MAG的基因疫苗免疫小鼠，小鼠的存活時(shí)間顯著延長(zhǎng)，且有8.3％的小鼠存活90天以上；以DNA疫苗初免、重組抗原疫苗加強(qiáng)免疫小鼠，33.3％的的小鼠存活90天以上。以上結(jié)果表明該疫苗具有良好的免疫保護(hù)性，驗(yàn)證了以多表位的方法制備弓形蟲(chóng)病疫苗、以復(fù)合免疫的方式增強(qiáng)免疫效果的可行性。 第二部分重組多表位抗原在免疫診斷中的初步應(yīng)用 1．弓形蟲(chóng)感染小鼠血清的制備及鑒定 以B36株弓形蟲(chóng)感染小鼠，采集血清，以ELISA和Western—blot檢測(cè)抗弓形蟲(chóng)IgG抗體，直接鏡檢腦內(nèi)包囊或腹腔速殖子，共獲得17只小鼠的感染血清，效價(jià)最高可達(dá)1：51200。 2．弓形蟲(chóng)多表位重組抗原的可溶性表達(dá)、純化及鑒定 大量誘導(dǎo)表達(dá)可溶性的弓形蟲(chóng)多表位重組抗原(rMAG)，以Ni-NTA agarose和割膠電滲對(duì)其進(jìn)行兩步法純化，最后得到了純度達(dá)95.86％的rMAG。免疫印跡實(shí)驗(yàn)證明，弓形蟲(chóng)多表位重組可溶性抗原不僅能夠被弓形蟲(chóng)強(qiáng)毒株RH感染的兔血清及抗弓形蟲(chóng)速殖子主要表面抗原SAG1(P30)的單抗所識(shí)別，而且能被成囊株弓形蟲(chóng)B36株慢性感染的小鼠血清所識(shí)別，提示該抗原既含有弓形蟲(chóng)速殖子抗原表位，也含有緩殖子包囊抗原表位。 3．弓形蟲(chóng)多表位重組抗原在檢測(cè)弓形蟲(chóng)感染中的應(yīng)用 以弓形蟲(chóng)感染血清為一抗，篩選重組弓形蟲(chóng)多表位抗原的最佳包被濃度為3μg／ml，用于制備ELISA檢測(cè)試劑盒。 檢測(cè)小鼠血清141份，其中陽(yáng)性小鼠感染血清為117份、正常小鼠血清24份，結(jié)果顯示，該試劑盒敏感性為88.88％(104／117×100％)，特異性為91.67％(22／24×100％)，一致性為89.36％((104+22)／141×100％)。 檢測(cè)兔血清24份，其中急性感染弓形蟲(chóng)的兔血清18份，正常兔血清6份，陽(yáng)性血清檢出率為94.4％，總一致性為91.5％((17+5)／24×100％)。 以上結(jié)果顯示，弓形蟲(chóng)多表位抗原試劑盒既可以檢測(cè)弓形蟲(chóng)急性感染血清，又可以檢測(cè)弓形蟲(chóng)慢性感染血清，初步證實(shí)了多表位抗原用于弓形蟲(chóng)感染診斷中的優(yōu)勢(shì)，為試劑盒的下一步應(yīng)用奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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