免疫途径、剂量和佐剂对CVB3VP1蛋白免疫效果的影响

发布时间：2018-06-19 18:56

本文选题：VP1 + 柯萨奇病毒B组3型(CVB3)　；参考：《河北医科大学》2009年硕士论文

【摘要】： 目的:柯萨奇病毒属于小RNA病毒科肠道病毒属。其中B组3型(Coxsackievirus group B type 3, CVB3)所致的病毒性心肌炎严重危害人类健康,也是儿童和青少年猝死的重要原因,迄今尚无有效的预防手段。VP1是CVB3主要的衣壳蛋白,含有多个T、B细胞表位,可诱导机体产生免疫应答。基因工程亚单位疫苗是将目的基因克隆到原核或真核表达载体中,在原核或真核细胞中表达,经纯化制备的疫苗。亚单位疫苗具有较强的免疫原性,成分单一,可显著提高抗体水平,免除非抗原成分引起的副作用,保证疫苗的安全性。本室前期将VP1基因克隆到原核表达载体,在大肠杆菌成功表达了VP1蛋白,经纯化后免疫小鼠。结果显示VP1蛋白能诱导产生较高水平的体液免疫和细胞免疫应答,但未能达到满意的保护效果。因此,进一步增强VP1蛋白的免疫原性,提高CVB3特异性体液和细胞免疫应答,已成为倍受关注的研究方向。有多种因素影响机体对抗原的免疫应答强度及类型,主要是抗原物质本身的性质和进入机体的途径、剂量、次数、免疫间隔时间以及免疫佐剂的类型等。本研究用原核细胞表达得到纯化CVB3VP1蛋白,选取3种常用免疫途径,3种免疫剂量,联合3种免疫佐剂共同免疫小鼠。观察不同免疫途径,不同剂量和不同佐剂对免疫效果的影响。方法: 1将我室构建的原核表达质粒pET-his/VP1转入表达宿主菌BL21(DE3)pLysS,诱导表达携带有6×HIS标签的VP1蛋白,经SDS-PAGE和Western-blot鉴定。 2将大量诱导表达后的菌体在冰浴中超声破碎,分离上清和包涵体沉淀,经SDS-PAGE分析,结果表明蛋白以包涵体形式存在,用尿素溶解沉淀,利用金属镍离子亲和层析柱纯化目的蛋白,透析后浓缩蛋白。 3动物实验:分二期进行。第一期将小鼠随机分为3组,每组12只,分别为腹腔注射组、皮下注射组、肌肉注射组。纯化后的蛋白以PBS稀释后,经过腹腔、颈背部皮下和股四头肌注射免疫小鼠。初次免疫加完全弗氏佐剂,以后免疫用不完全弗氏佐剂。每次每只接种100μg;每3周免疫1次,共免疫3次;每次免疫后第14天,内眦静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)检测血清中CVB3特异性中和抗体的水平;用纯化VP1抗原包被酶联板,采用ELISA方法检测小鼠血清抗体IgG抗体效价。经过比较选择最佳免疫注射途径,进行第二期动物实验。 4第二期动物实验:将6～8周龄雄性BALB/c小鼠随机分为6组,每组18只。纯化后的蛋白以PBS稀释后,采用肌肉注射途径免疫,①低剂量组:每次每只接种50μg。②中剂量组:每次每只接种100μg。③高剂量组:每次每只接种150μg。以上3组初次免疫加完全弗氏佐剂,以后免疫用不完全弗氏佐剂。④氢氧化铝佐剂组:每次每只接种100μg。⑤ISA 720组:每次每只接种100μg。⑥PBS组。每3周免疫1次,共免疫3次;每次免疫后第14天,内眦静脉取血,分离血清,用微量中和试验(固定病毒-稀释血清法)检测血清中CVB3特异性中和抗体的水平;用纯化VP1抗原包被酶联板,采用ELISA方法检测小鼠血清特异性抗体效价。第3次免疫后3周,每组3只小鼠取脾脏制备淋巴细胞悬液,采用细胞计数试剂盒(cell counting kit-8, CCK-8)法进行特异性淋巴细胞增殖活性和特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte, CTL)杀伤活性的检测;另每组12只小鼠用致死量的CVB3(4LD_(50))进行腹腔注射,观察并记录小鼠死亡情况至感染后第21天;每组剩余的3只小鼠用CVB3(3LD_(50))攻击,注射病毒后第7天处死,用于血中病毒滴度的测定。结果: 1 SDS-PAGE和Western-blot证实原核表达质粒pET-his/VP1转化的大肠杆菌BL21(DE3)pLysS成功表达了CVB3 VP1蛋白,蛋白主要以包涵体形式存在。 2不同免疫途径各组各次免疫后血清中和抗体滴度分别为:腹腔注射组: 1:7.08,1:28.18,1:42.66;皮下注射组:1:7.08,1:11.22,1:35.48;肌肉注射组:1: 8.91,1:47.86,1:70.79。血清特异性IgG抗体分别为:皮下注射组:1:155,1: 7600,1:11800;腹腔注射组1:240, 1:8300,1:17000;肌肉注射组1:425,1:22400,1:38400。单因素方差分析表明,三次免疫后,各组中和抗体和特异性IgG抗体滴度逐次增加(P0.05)。三次免疫后,中和抗体和特异性IgG抗体:肌肉注射组腹腔注射组皮下注射组(P0.01)。 3经肌肉注射途径免疫后,不同剂量各组血清中和抗体效价分别为:低剂量组:1:6.31, 1:42.66, 1:47.86;中剂量组:1:7.08, 1:44.67, 1:63.10;高剂量组: 1:7.08, 1:50.12,1:70.79。血清特异性IgG水平:低剂量组: 1:200, 1:6400, 1: 19200;中剂量组: 1:398, 1:21000, 1:36300;高剂量组: 1:425, 1:23040, 1:51200。单因素方差分析表明,三次免疫后,各组中和抗体和特异性IgG抗体滴度逐次增加(P0.05)。末次免疫后各组比较差别有统计学意义。中和抗体和特异性IgG抗体:高剂量组中剂量组低剂量组(P0.05)。 4不同佐剂组各次免疫后血清中和抗体滴度为:氢氧化铝佐剂组: 1:5.62, 1:12.59, 1:26.92;弗氏佐剂(中剂量)组: 1:7.08, 1:44.67, 1:63.10; ISA720佐剂组: 1:8.91, 1: 44.67, 1: 85.11。各次免疫后血清特异性IgG水平:氢氧化铝佐剂组: 1:150, 1:1600,1:11800;弗氏佐剂(中剂量)组:1:398, 1:21000, 1:36300; ISA720佐剂组:1:280, 1:12800,1:38400。单因素方差分析表明,三次免疫后,各组中和抗体和特异性IgG抗体滴度逐次增加(P0.05)。末次免疫后各组比较差别有统计学意义。中和抗体:ISA720佐剂组弗氏佐剂组氢氧化铝佐剂组(P0.05)。特异性IgG抗体:ISA720组和弗氏佐剂组高于氢氧化铝佐剂组,(P0.05)弗氏佐剂组和ISA720组差别无统计学意义(P0.05)。 5小鼠脾脏淋巴细胞增殖活性,以CVB3或Con A刺激,各组均高于PBS组(P0.05);以CVB3刺激,高剂量组高于低剂量组和中剂量组(P0.05); ISA720佐剂组高于弗氏佐剂组和氢氧化铝佐剂组(P0.05)。ConA刺激时,各组均显著高于PBS组(P0.05)。各组之间差别无统计学意义。 6小鼠脾脏特异性CTL杀伤活性,各组均显著高于PBS组(P0.05);高剂量组高于中剂量组和低剂量组(P0.05);弗氏佐剂组和ISA720组高于氢氧化铝组(P0.05)。 7用4LD_(50)攻击小鼠,PBS组、低剂量组、中剂量组、高剂量组、氢氧化铝佐剂组、ISA720佐剂组的21天生存率分别为: 0、66.67%、66.67%、50%、33.33%和50 %。经χ2检验分析,各组之间无明显差异(P0.05)。经Kaplan-Meier法进行生存分析表明,各组生存率曲线分布有差别(P0.05)。低剂量组和中剂量组的生存时间好于高剂量组,弗氏佐剂组生存时间好于氢氧化铝佐剂组(P0.05)。 8各组血清病毒滴度均比PBS组低(P0.05)。单因素方差分析显示,高剂量组血中病毒滴度低于低、中剂量组(P0.05);ISA720佐剂组低于氢氧化铝组(P0.05)。结论: 1成功在原核细胞表达了CVB3 VP1蛋白,并进行了纯化。 2三种免疫途径接种,各组血清中和抗体和IgG抗体滴度均随免疫次数的增加而提高,肌肉免疫能诱导更高的体液免疫水平。 3采用肌肉注射途径免疫,中和抗体和IgG抗体水平随免疫剂量增加而提高。用致死量病毒攻击后,病毒载量随免疫剂量增加而降低,但低剂量组的生存时间好于其他剂量组。 4三种佐剂比较,弗氏佐剂和ISA 720佐剂的对VP1蛋白的免疫增强作用优于氢氧化铝佐剂。但是弗氏佐剂不能应用于人,因此ISA 720佐剂是一种值得进一步研究的有效佐剂。 5采用合适剂量(50-100μg /只)的VP1蛋白,应用弗氏佐剂或ISA 720佐剂,选择肌肉注射途径免疫可获得较好的免疫效果。
[Abstract]:Objective: Coxsackie virus belongs to the enterovirus of small RNA virus. Viral myocarditis caused by B group 3 (Coxsackievirus group B type 3, CVB3) is a serious cause of human health. It is also an important cause of sudden death in children and adolescents. So far, there is no effective prevention method,.VP1 is the main capsid protein of CVB3, containing multiple T, B cell table. It can induce immune response in the body.
The subunit vaccine of gene engineering is to clone the target gene into the prokaryotic or eukaryotic expression vector, expressed in the prokaryotic or eukaryotic cells, and the purified vaccine. The subunit vaccine has a strong immunogenicity and a single component. It can significantly improve the antibody level, avoid the side effects caused by non antigen components and ensure the safety of the vaccine. The VP1 gene was cloned into the prokaryotic expression vector in the early stage. The VP1 protein was successfully expressed in Escherichia coli and immunized in mice. The results showed that the VP1 protein could induce a high level of humoral immunity and cellular immune response, but failed to achieve a satisfactory protective effect. Therefore, the immunogenicity of the VP1 protein was enhanced and the specificity of the CVB3 was enhanced. Humoral and cellular immune responses have attracted much attention.
There are many factors affecting the immune response intensity and type of the body, mainly the nature of the antigen and the way to enter the body, the dose, the number of times, the time of the immune interval, and the type of the immune adjuvant. This study uses the expression of the prokaryotic cells to purify the CVB3VP1 protein, select 3 kinds of common immunization routes and 3 kinds of immune doses. 3 immune adjuvants were used to immunize mice. The effects of different routes of immunization, different doses and different adjuvants on immune response were observed.
Method:
1 the prokaryotic expression plasmid pET-his/VP1 constructed in our room was transferred into the expression host bacterium BL21 (DE3) pLysS, and the expression of VP1 protein with a 6 x HIS label was induced and identified by SDS-PAGE and Western-blot.
2 the bacteria after a large number of induced expression were broken in the ice bath, separating the supernatant and inclusion body precipitation. After SDS-PAGE analysis, the results showed that the protein existed in the form of inclusion body, dissolved in urea and precipitated, purified the target protein by metal nickel ion affinity chromatography column, and after dialysis.
3 animal experiments were carried out in two stages. In the first stage, the mice were randomly divided into 3 groups, 12 in each group, which were intraperitoneal injection group, subcutaneous injection group and intramuscular injection group. After the purified protein was diluted by PBS, the mice were vaccinated through the abdominal cavity, the neck back subcutaneous and the four head of the femoral head. The first immunization and complete Freund's adjuvant were used for the later immunization. Each inoculant was inoculated 100 mu g each time, immunized 1 times every 3 weeks, 3 times, and fourteenth days after each immunization, the inner canthus vein was taken blood, the serum was separated, the level of CVB3 specific neutralization antibody in serum was detected by microneutralization test (fixed virus dilution sera); the serum antibody IgG was detected by the purified VP1 antigen, and the ELISA method was used to detect the anti IgG anti serum antibody of mice. The best immunization route was selected for the second phase of animal experiment.
4 second phase of animal experiment: 6~8 weeks male BALB/c mice were randomly divided into 6 groups, each group was 18. After the purified protein was diluted by PBS, the mice were immunized by intramuscular injection. (1) the low dose group: each time each inoculation was 50 mu g.. Each time was inoculated 100 mu G. high dose group: each inoculated more than 3 groups of 3 groups above 3 groups for the first time. The whole Freudian adjuvant, after immunization with incomplete Freund adjuvant. (4) group of aluminum hydroxide adjuvant: each group was inoculated 100 mu g. 5 ISA 720 groups each time, each inoculated with 100 mu g. 6 PBS groups. Every 3 weeks immunization 1 times, CO immunization was 3 times, and fourteenth days after each immunization, the inner canthus vein was blood, separated serum, microneutralization test (fixed virus dilution sera) test The level of CVB3 specific neutralization antibody in the serum, the purified VP1 antigen was coated with the ELISA and the ELISA method was used to detect the titer of the specific antibody in the mice serum. 3 weeks after third immunizations, 3 mice in each group were prepared with the spleen to prepare the lymphocyte suspension, and the cell count Kit (cell counting kit-8, CCK-8) was used for the specific lymphocyte proliferation. The activity and specific cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) killing activity was detected; another 12 mice in each group were intraperitoneally injected with lethal CVB3 (4LD_ (50)), observed and recorded the death of mice to twenty-first days after infection; the remaining 3 mice in each group were attacked with CVB3 (3LD_ (50)), and were killed seventh days after the injection of the virus. Determination of the titer of the virus in blood.
Result:
1 SDS-PAGE and Western-blot confirmed that the Escherichia coli BL21 (DE3) pLysS transformed by the prokaryotic expression plasmid pET-his/VP1 successfully expressed the CVB3 VP1 protein, and the protein was mainly in the form of inclusion body.
2 the serum neutralizing antibody titers of each immunization group were: intraperitoneal injection group: 1:7.08,1:28.18,1:42.66; subcutaneous injection group: 1:7.08,1:11.22,1:35.48; intramuscular injection group: 1: 8.91,1:47.86,1:70.79. serum specific IgG antibodies were: subcutaneous injection group: 1:155,1: 7600,1:11800; intraperitoneal injection of 1:240, 1:8300,1:170 00, the single factor analysis of variance of 1:425,1:22400,1:38400. in the intramuscular injection group showed that the titer of neutralizing antibody and specific IgG antibody increased after three times of immunization (P0.05). After three times of immunization, neutralizing antibody and specific IgG antibody: intraperitoneal injection group subcutaneous injection group (P0.01).
3 after immunization by intramuscular injection, the titer of serum neutralization antibody in different doses were: low dose group: 1:6.31, 1:42.66, 1:47.86; middle dose group: 1:7.08, 1:44.67, 1:63.10; high dose group: 1:7.08, 1:50.12,1:70.79. serum specific IgG level: low dose group: 1:200, 1:6400, 1: 19200; medium dose group; medium dose group; High dose group: single factor analysis of variance of 1:425, 1:23040 and 1:51200. showed that the titer of neutralizing antibody and specific IgG antibody increased after three times of immunization (P0.05). The difference was statistically significant after the last immunization. Neutralization antibody and specific IgG antibody: low dose group (P0.05) in high dose group.
4 the serum neutralizing antibody titers of different adjuvant groups were: 1:5.62, 1:12.59, 1:26.92, Freund's adjuvant (middle dose) group: 1:7.08, 1:44.67, 1:63.10; ISA720 adjuvant group: 1:8.91, 1: 44.67, 1: 85.11., serum specific IgG levels: aluminum hydroxide adjuvant group: Ephesians Group (medium dose) group: 1:398, 1:21000, 1:36300 and ISA720 adjuvant group: 1:280, 1:12800,1:38400. single factor analysis of variance showed that the neutralization antibody and specific IgG antibody titer increased successive (P0.05) after three times of immunization. The comparison of each group after the last immunization was significant. Neutralization antibody: the Freund's adjuvant group of ISA720 adjuvant group The adjuvant group (P0.05). Specific IgG antibody: the group ISA720 and the Freund adjuvant group were higher than the aluminum hydroxide adjuvant group. (P0.05) the difference between the Freund's adjuvant group and the ISA720 group was not statistically significant (P0.05).
5 mice spleen lymphocyte proliferation activity was stimulated by CVB3 or Con A, all groups were higher than group PBS (P0.05), with CVB3 stimulation, high dose group was higher than low dose group and middle dose group (P0.05); ISA720 adjuvant group was higher than that of Freund's adjuvant group and aluminum hydroxide adjuvant group (P0.05).ConA stimulation, all groups were significantly higher than PBS group (P0.05). The difference between each group was not unified. The significance of learning.
6 the killing activity of spleen specific CTL in mice was significantly higher than that in PBS group (P0.05), the high dose group was higher than the middle dose group and low dose group (P0.05), and the Freund and ISA720 group were higher than the aluminum hydroxide group (P0.05).
7 using 4LD_ (50) to attack mice, group PBS, low dose group, middle dose group, high dose group, aluminum hydroxide adjuvant group and ISA720 adjuvant group, the 21 natural survival rates were 0,66.67%, 66.67%, 50%, 33.33% and 50. There was no significant difference between each group by chi square test (P0.05). The survival analysis showed that the survival rate curves of each group were distributed by the Kaplan-Meier method. Difference (P0.05). The survival time of low dose group and middle dose group was better than that of high dose group, and the survival time of Freund's adjuvant group was better than that of aluminum hydroxide adjuvant group (P0.05).
8 the titer of serum virus in all groups was lower than that of the PBS group (P0.05). The single factor analysis of variance showed that the virus titer in the high dose group was lower than that of the low dose group (P0.05), and the ISA720 adjuvant group was lower than the aluminum hydroxide group (P0.05).
Conclusion:
1 CVB3 VP1 protein was successfully expressed in prokaryotic cells and purified.
2 three kinds of immunization were inoculated. The serum neutralization antibody and the titer of IgG antibody were increased with the increase of the number of immunization. The muscle immunity could induce a higher level of humoral immunity.
3 immunization by intramuscular injection, the level of neutralizing antibody and IgG antibody increased with the increase of immune dose. After the attack of lethal virus, the viral load decreased with the increase of immune dose, but the survival time of the low dose group was better than that of the other dose groups.
4 compared with three kinds of adjuvant, the immune enhancement effect of Freund's adjuvant and ISA 720 adjuvant on VP1 protein is better than that of aluminum hydroxide adjuvant. However, Freund's adjuvant can not be used in human beings, so ISA 720 adjuvant is an effective adjuvant for further study.
5 using the appropriate dose (50-100 g / VP1) of the VP1 protein and using Freund's adjuvant or ISA 720 adjuvant to select the intramuscular injection, the immunization effect will be better.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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