Linker长度对MDC和CVB3VP1融合基因疫苗免疫效果的影响

发布时间：2018-01-05 04:08

本文关键词：Linker长度对MDC和CVB3VP1融合基因疫苗免疫效果的影响　出处：《河北医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:柯萨奇病毒B组3型(Coxsackievirus group B type 3, CVB3)是导致人类急、慢性心肌病的重要病原一。其预防和治疗性疫苗的研究极为迫切。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。国内外研究表明,编码VP1的DNA疫苗,能诱导小鼠产生体液和细胞免疫应答,并对感染小鼠具有一定的保护能力,但不能有效阻止致死量病毒感染。研究证明,一些细胞因子作为基因佐剂,可大大提高DNA疫苗的免疫效应。本室构建了MDC ( macrophage-derived chemokine, MDC )与CVB3VP1融合基因质粒,保护率有一定的提高,但不能达到实用程度。推测可能是融合蛋白中某一成分或两种成分发生空间结构的不利变化,影响与APCs结合和/或VP1蛋白的免疫原性。如何保证融合蛋白的稳定性和生物学活性是本研究探讨的问题。接头序列(Linker)的介入是基因融合技术之一,即通过一段适当的核苷酸序列将不同的目的基因连接起来,使之在适当的生物体内表达成为一条单一的肽链,其中起连接作用的氨基酸称为Linker。Linker的选择对构建有功能活性的融合蛋白是至关重要的。它的存在有利于蛋白质两侧功能区的正确折叠,以允许形成各自独立的构象。长度和组成是选择Linker时主要应考虑的问题。研究表明,亲水性强和柔性好的Linker是构建融合蛋白的首选。故甘氨酸(glycine, Gly, G)和丝氨酸(serine, Ser, S)是构建Linker的常用氨基酸。G是所有氨基酸中分子量最小,没有手性碳,所以柔性最好,S是亲水性最强的氨基酸,能增加亲水性。Linker的长度过长,在融合蛋白的生产过程中对蛋白酶比较敏感,导致活性融合蛋白的产量减低,应用较短的Linker,可以克服重组蛋白酶分解的问题,但可能使两个分子距离太近导致蛋白功能的丧失。据资料显示10-22个氨基酸效果较好。故本研究引入长度为10、15、19个由G和S组成的Linker来构建表达MDC与CVB3VP1融合蛋白核酸疫苗。方法:(1)用PCR方法分别扩增出带Linker的MDC-L15(用于构建Linker长度为15个氨基酸的融合蛋白)、MDC-L19(用于构建Linker长度为19个氨基酸的融合蛋白)、L15-VP1(用于构建Linker长度为15个氨基酸的融合蛋白)和L19-VP1(用于构建Linker长度为19个氨基酸的融合蛋白)片段;(2)将4种基因的扩增产物与pGEM-T载体连接,转化DH5α大肠杆菌,接种含Amp的2YT培养基,提取质粒进行酶切鉴定和测序筛选重组子;(3)经酶切鉴定和测序确认后,用合适的核酸内切酶从这4种克隆载体切取目的基因片段。将目的基因片段MDC-L15和L15-VP1与经合适限制性内切酶酶切的pcDNA3载体连接,构建真核表达重组质粒pcDNA3/MDC-L15-VP1。同样的方法构建pcDNA3/ MDC-L19-VP1。(4)用碱裂解法从转化的DH5α大肠杆菌中大量提取质粒pcDNA3/MDC-L19-VP1、pcDNA3/MDC- L15- VP1、pcDNA3/MDC-L10-VP1、pcDNA3/VP1和pcDNA3,用聚乙二醇(PEG)沉淀法进行纯化;(5)动物实验:将6周龄雄性BALB/c小鼠随机分为5组,每组14只;5组分别为pcDNA3组、pcDNA3/VP1组、pcDNA3/MDC-L10-VP1组、pcDNA3/MDC-L15-VP1组和pcDNA3/MDC-L19-VP1组。纯化后的质粒以生理盐水稀释后,股四头肌注射免疫小鼠,每次每只接种100μg,每4周免疫1次,共免疫3次;于2、6、10周,眼眶静脉取血,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。第3次免疫后3周,每组3只小鼠取脾脏用于检测细胞免疫水平,每组8只小鼠用5 LD50的CVB3腹腔内攻击,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3 LD50CVB3攻击,注射病毒后第7天取血,分离血清进行病毒滴度测定。处死后,取心脏,制备石蜡切片,HE染色,观察各组小鼠心肌病理学改变。结果:(1) PCR扩增出MDC-L15、MDC-L19、L15-VP1和L19-VP1基因片段,基因片段的长度与预期值相符。(2)成功构建了原核克隆载体pGEM-T/MDC-L15,pGEM-T /MDC-L19, pGEM-T/L15-VP1和pGEM-T/L19 -VP1, DNA测序证实目的基因序列与Genbank登录序列相同。(3)基因片段插入pcDNA3后酶切结果证实成功构建了pcDNA3/MDC-L15-VP1和pcDNA3/MDC-L-19-VP1。(4)不同免疫次数,各组血清中和抗体的平均效价分别为:pcDNA3/VP1组1:7.07,1:10.59,1:25.20;pcDNA3/MDC- L10-VP1组1:7.93,1:22.45,1:31.74; pcDNA3/MDC-L15-V P1组1:8.91,1:23.78,1:39.99;pcDNA3/MDC-L9-VP1组1:10.00,1:25.20,1:50.39;pcDNA3组各次平均效价均低于1:5。pcDNA3/VP1、pcDNA3/MDC-L10-VP1、pcDNA3/MDC-L15-VP1和pcDNA3/MDC-L19-VP1组中和抗体滴度随免疫次数增加而提高,经单因素方差分析差别有统计学意义(P0.01);第三次免疫后,除pcDNA3/VP1组和pcDNA3/MDC-L10-VP1组无显著性差异,其余各组两两比较均有显著性差异(P 0.01)。(5)特异性淋巴细胞增殖实验和CTL杀伤实验结果表明,随Linker长度的增加增殖能力和杀伤活性显著增强,pcDNA3/MDC-L19-VP1组和其它各组相比差别均有统计学意义(P0.01)。(6)用5LD50攻击小鼠,各组21天生存率分别为:pcDNA3/MDC-L-15-VP1组和pcDNA3/MDC-L19-VP1组均为37.5%,pcDNA3/MDC-L10- VP1组25.0%,pcDNA3/VP1组12.5%, pcDNA3组11天内全部死亡。(7)随Linker长度的增加血中病毒滴度依次降低, pcDNA3/MDC-L19-VP1组小鼠和其它各组相比差别均有统计学意义。(8)心肌病理学检查随Linker长度的增加心肌病理改变依次减轻,但并无明显异。结论:(1)用PCR方法成功扩增MDC-L15、MDC-L19、L15-VP1和L19-VP1基因片段。(2)成功构建真核表达质粒pcDNA3/MDC-L15-VP1、pcDNA3/MDC-L19-VP1。(3)用5LD50的CVB3攻击后,小鼠的生存率随着Linker长度的加长有一定的提高。(4)小鼠血清中和抗体滴度随免疫次数的增加而提高,第三次免疫后pcDNA3/MDC-L19-VP1组中和抗体滴度最高,与其它各组相比差异均有统计学意义。(5)特异性淋巴细胞增殖实验和CTL杀伤实验结果表明pcDNA3/MDC-L19-VP1组增殖指数和杀伤率最高,与其它各组相比差异均有统计学意义。(6)中和抗体滴度、细胞免疫检测、血中病毒滴度、和心肌切片病理学分析结果一致,说明融合基因疫苗pcDNA3/MDC-L19-VP1是较理想的一种疫苗。
[Abstract]:Objective: coxsackievirus B 3 (Coxsackievirus group B type 3, CVB3) is an important pathogen causing human acute and chronic cardiomyopathy. The prevention and treatment of AIDS vaccine research is extremely urgent.VP1 is a major capsid protein of CVB3 contains a number of T, B cell epitope can induce immune response. The domestic and foreign research shows that VP1 encoding DNA vaccine can induce the humoral and cellular immune responses induced by mice, and has a protective ability of infected mice, but can not effectively prevent lethal virus infection. Studies have shown that some cytokines as an adjuvant, can greatly improve the immune effect of DNA vaccine. The room construction MDC (macrophage-derived chemokine MDC) and CVB3VP1 fusion gene plasmid, the protection rate increased to a certain extent, but can not reach the practical level. That is a component of a fusion protein or two components of space The unfavorable changes of structure affect the immunogenicity of APCs binding and / or VP1 protein. How to ensure the stability and biological activity of fusion protein is the problem discussed in this study.
Joint sequence (Linker) is one of the intervention of gene fusion technology, through an appropriate nucleotide sequence linking different target gene, making it a single peptide expressed in appropriate organisms, including the amino acid called Linker.Linker to construct a functional activity of the fusion protein was essential. It is conducive to the correct folding of protein function area on both sides, to allow the formation of conformation independent. The length and composition is mainly the choice of Linker should be considered. The results show that strong hydrophilicity and flexibility of Linker is to construct the fusion protein. The preferred glycine (Glycine, Gly, G) serine (serine, Ser, and S) is a common amino acid.G to construct the Linker molecules of all amino acids in the minimum amount, no chiral carbon, so the best S is the most flexible, hydrophilic amino acid, can To increase the hydrophilicity of.Linker length is too long, in the production process of fusion protein of protease sensitive activity in the fusion protein yield decreased by short Linker, can overcome the recombinant protease decomposition problem, but may make two molecules too close to lead to the loss of protein function. According to the data shows good 10-22 amino acids the purpose of this study is introduced. The effect of length 10,15,19 is composed of G and S Linker to construct the MDC expression of CVB3VP1 fusion protein and nucleic acid vaccine.
Methods: (1) were amplified by PCR with Linker MDC-L15 (Linker for the construction of a length of 15 amino acid fusion protein (MDC-L19), Linker is used to build the length of 19 amino acid fusion protein (L15-VP1), Linker is used to build the length of 15 amino acid fusion protein (L19-VP1) and used to construct Linker the length of 19 amino acid fragment fusion protein); (2) the amplified products were connected with pGEM-T vector 4 genes, transformation of Escherichia coli DH5 alpha, inoculation with Amp 2YT medium, extraction of plasmid enzyme digestion and sequencing screening recombinant; (3) confirmed by enzyme digestion and sequencing after that cut the target gene fragment from the 4 kinds of cloning vector by endonuclease appropriate. The target gene fragments of MDC-L15 and L15-VP1 and the suitable vector pcDNA3 restriction endonuclease connection, construct eukaryotic expression recombinant plasmid pcDNA3/MDC-L15-VP1. the same way Construction of pcDNA3/ MDC-L19-VP1. (4) extracting plasmid pcDNA3/MDC-L19-VP1 from Escherichia coli DH5 alpha conversion by alkaline lysis method, pcDNA3/MDC- L15- VP1, pcDNA3/MDC-L10-VP1, pcDNA3/VP1 and pcDNA3, using polyethylene glycol (PEG) was purified by precipitation method; (5) animal experiment: 6 week old male BALB/c mice were randomly divided into 5 groups, each group 14; 5 groups were divided into pcDNA3 group, pcDNA3/VP1 group, pcDNA3/MDC-L10-VP1 group, pcDNA3/MDC-L15-VP1 group and pcDNA3/MDC-L19-VP1 group. The purified plasmid was diluted with saline after femoral head four muscle injection of immune mice, each with 100 g, 1 times of immunization every 4 weeks, a total of 3 times of immunization in 2,6,10 weeks,; orbital venous blood, serum separation, micro neutralization test (fixed virus serum dilution method) to detect serum CVB3 specific neutralizing antibodies. Third 3 weeks after immunization, 3 mice in each group were used to detect the cellular immunity of the spleen, n = 8 Mice with 5 LD50 CVB3 intraperitoneal attack, observe and record the death of mice to twenty-first days after infection. 3 mice in each group with the remaining 3 LD50CVB3 attack, blood samples were collected seventh days after infected, the virus titer of the serum were separated from heart. After the execution, and the preparation of paraffin section, HE staining, observation study the pathological changes of myocardium in mice.
Results: (1) PCR amplified MDC-L15, MDC-L19, L15-VP1 and L19-VP1 gene fragments, and the expected length gene fragment valuescorrespond. (2) successfully constructed prokaryotic cloning vector pGEM-T/MDC-L15, pGEM-T /MDC-L19, pGEM-T/L15-VP1 pGEM-T/L19 and -VP1 DNA sequencing confirmed that the gene sequence and the Genbank sequence of the same log. (3) gene fragment insert pcDNA3 after enzyme digestion confirmed the successful construction of pcDNA3/MDC-L15-VP1 and pcDNA3/MDC-L-19-VP1. (4) different immune times, the average titer of neutralizing antibody in serum were respectively: group pcDNA3/VP1 1:7.07,1: 10.59,1:25.20; pcDNA3/MDC- L10-VP1 1:7.93,1:22.45,1:31.74 pcDNA3/MDC-L15-V P1 group; 1:8.91,1:23.78,1:39.99 group; pcDNA3/MDC-L9-VP1 1:10.00,1:25.20,1:50.39 group; pcDNA3 group the average titer were lower than those of 1:5.pcDNA3/VP1, pcDNA3/MDC-L10-VP1, pcDNA3/MDC-L15-VP1 and pcDNA3/MDC-L19-VP1 the titer of neutralizing antibody group To increase the number of immune, the single factor analysis of variance the difference was statistically significant (P0.01); after the third immunization, except pcDNA3/VP1 group and pcDNA3/MDC-L10-VP1 group had no significant difference between the 22 groups, there were significant differences (P 0.01). (5) the specific lymphocyte proliferation assay and CTL cytotoxicity assay results show that with the increase of Linker length proliferation and killing activity was significantly enhanced, compared the difference of both pcDNA3/MDC-L19-VP1 group and other groups (P0.01). (6) the mice were challenged with 5LD50, the survival rate of 21 groups respectively: pcDNA3/MDC-L-15-VP1 group and pcDNA3/MDC-L19-VP1 group were 37.5%, pcDNA3/MDC-L10- and 25% in VP1 group, pcDNA3/VP1 group of 12.5%. The pcDNA3 group all died within 11 days. (7) with the increase of Linker length in order to reduce the virus titer in the blood group pcDNA3/MDC-L19-VP1, mice and other groups showed statistical differences. (8) myocardial pathology examination decreased in turn with the increase of Linker length, but there was no significant difference.
Conclusion: (1) were successfully amplified by PCR MDC-L15, MDC-L19, L15-VP1 and L19-VP1 gene fragments. (2) the eukaryotic expression plasmid pcDNA3/MDC-L15-VP1 was successfully constructed, pcDNA3/MDC-L19-VP1. (3) by 5LD50 CVB3 after the attack, the survival rate of mice with Linker length extension has improved to some extent. (4) serum neutralizing antibody titer with the times of immunization increased, after the third immunization group pcDNA3/MDC-L19-VP1 neutralizing antibody titers, compared with that of the other groups were statistically significant. (5) the specific lymphocyte proliferation assay and CTL cytotoxicity test results showed that the proliferation index in pcDNA3/MDC-L19-VP1 group and the killing rate was the highest compared with other groups, the differences were statistically significant (6). The titer of neutralizing antibody, immune detection, virus titer in the blood and myocardial pathological section analysis showed that this fusion gene vaccine pcDNA3/MDC-L19-VP1 is ideal A vaccine.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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