本文關鍵詞：Linker長度對MDC和CVB3VP1融合基因疫苗免疫效果的影響　出處：《河北醫(yī)科大學》2007年碩士論文　論文類型：學位論文

  更多相關文章： 巨噬細胞源趨化因子(MDC) 柯薩奇病毒B組3型(CVB3) Linker 核酸疫苗 中和抗體 小鼠

【摘要】： 目的:柯薩奇病毒B組3型(Coxsackievirus group B type 3, CVB3)是導致人類急、慢性心肌病的重要病原一。其預防和治療性疫苗的研究極為迫切。VP1是CVB3主要的衣殼蛋白,含有多個T、B細胞表位,可誘導機體產生免疫應答。國內外研究表明,編碼VP1的DNA疫苗,能誘導小鼠產生體液和細胞免疫應答,并對感染小鼠具有一定的保護能力,但不能有效阻止致死量病毒感染。研究證明,一些細胞因子作為基因佐劑,可大大提高DNA疫苗的免疫效應。本室構建了MDC ( macrophage-derived chemokine, MDC )與CVB3VP1融合基因質粒,保護率有一定的提高,但不能達到實用程度。推測可能是融合蛋白中某一成分或兩種成分發(fā)生空間結構的不利變化,影響與APCs結合和/或VP1蛋白的免疫原性。如何保證融合蛋白的穩(wěn)定性和生物學活性是本研究探討的問題。 接頭序列(Linker)的介入是基因融合技術之一,即通過一段適當?shù)暮塑账嵝蛄袑⒉煌哪康幕蜻B接起來,使之在適當?shù)纳矬w內表達成為一條單一的肽鏈,其中起連接作用的氨基酸稱為Linker。Linker的選擇對構建有功能活性的融合蛋白是至關重要的。它的存在有利于蛋白質兩側功能區(qū)的正確折疊,以允許形成各自獨立的構象。長度和組成是選擇Linker時主要應考慮的問題。研究表明,親水性強和柔性好的Linker是構建融合蛋白的首選。故甘氨酸(glycine, Gly, G)和絲氨酸(serine, Ser, S)是構建Linker的常用氨基酸。G是所有氨基酸中分子量最小,沒有手性碳,所以柔性最好,S是親水性最強的氨基酸,能增加親水性。Linker的長度過長,在融合蛋白的生產過程中對蛋白酶比較敏感,導致活性融合蛋白的產量減低,應用較短的Linker,可以克服重組蛋白酶分解的問題,但可能使兩個分子距離太近導致蛋白功能的喪失。據(jù)資料顯示10-22個氨基酸效果較好。故本研究引入長度為10、15、19個由G和S組成的Linker來構建表達MDC與CVB3VP1融合蛋白核酸疫苗。 方法:(1)用PCR方法分別擴增出帶Linker的MDC-L15(用于構建Linker長度為15個氨基酸的融合蛋白)、MDC-L19(用于構建Linker長度為19個氨基酸的融合蛋白)、L15-VP1(用于構建Linker長度為15個氨基酸的融合蛋白)和L19-VP1(用于構建Linker長度為19個氨基酸的融合蛋白)片段;(2)將4種基因的擴增產物與pGEM-T載體連接,轉化DH5α大腸桿菌,接種含Amp的2YT培養(yǎng)基,提取質粒進行酶切鑒定和測序篩選重組子;(3)經酶切鑒定和測序確認后,用合適的核酸內切酶從這4種克隆載體切取目的基因片段。將目的基因片段MDC-L15和L15-VP1與經合適限制性內切酶酶切的pcDNA3載體連接,構建真核表達重組質粒pcDNA3/MDC-L15-VP1。同樣的方法構建pcDNA3/ MDC-L19-VP1。(4)用堿裂解法從轉化的DH5α大腸桿菌中大量提取質粒pcDNA3/MDC-L19-VP1、pcDNA3/MDC- L15- VP1、pcDNA3/MDC-L10-VP1、pcDNA3/VP1和pcDNA3,用聚乙二醇(PEG)沉淀法進行純化;(5)動物實驗:將6周齡雄性BALB/c小鼠隨機分為5組,每組14只;5組分別為pcDNA3組、pcDNA3/VP1組、pcDNA3/MDC-L10-VP1組、pcDNA3/MDC-L15-VP1組和pcDNA3/MDC-L19-VP1組。純化后的質粒以生理鹽水稀釋后,股四頭肌注射免疫小鼠,每次每只接種100μg,每4周免疫1次,共免疫3次;于2、6、10周,眼眶靜脈取血,分離血清,用微量中和試驗(固定病毒-稀釋血清法)檢測血清中CVB3特異性中和抗體。第3次免疫后3周,每組3只小鼠取脾臟用于檢測細胞免疫水平,每組8只小鼠用5 LD50的CVB3腹腔內攻擊,觀察并記錄小鼠死亡情況至感染后第21天。每組剩余的3只小鼠用3 LD50CVB3攻擊,注射病毒后第7天取血,分離血清進行病毒滴度測定。處死后,取心臟,制備石蠟切片,HE染色,觀察各組小鼠心肌病理學改變。 結果:(1) PCR擴增出MDC-L15、MDC-L19、L15-VP1和L19-VP1基因片段,基因片段的長度與預期值相符。(2)成功構建了原核克隆載體pGEM-T/MDC-L15,pGEM-T /MDC-L19, pGEM-T/L15-VP1和pGEM-T/L19 -VP1, DNA測序證實目的基因序列與Genbank登錄序列相同。(3)基因片段插入pcDNA3后酶切結果證實成功構建了pcDNA3/MDC-L15-VP1和pcDNA3/MDC-L-19-VP1。(4)不同免疫次數(shù),各組血清中和抗體的平均效價分別為:pcDNA3/VP1組1:7.07,1:10.59,1:25.20;pcDNA3/MDC- L10-VP1組1:7.93,1:22.45,1:31.74; pcDNA3/MDC-L15-V P1組1:8.91,1:23.78,1:39.99;pcDNA3/MDC-L9-VP1組1:10.00,1:25.20,1:50.39;pcDNA3組各次平均效價均低于1:5。pcDNA3/VP1、pcDNA3/MDC-L10-VP1、pcDNA3/MDC-L15-VP1和pcDNA3/MDC-L19-VP1組中和抗體滴度隨免疫次數(shù)增加而提高,經單因素方差分析差別有統(tǒng)計學意義(P0.01);第三次免疫后,除pcDNA3/VP1組和pcDNA3/MDC-L10-VP1組無顯著性差異,其余各組兩兩比較均有顯著性差異(P 0.01)。(5)特異性淋巴細胞增殖實驗和CTL殺傷實驗結果表明,隨Linker長度的增加增殖能力和殺傷活性顯著增強,pcDNA3/MDC-L19-VP1組和其它各組相比差別均有統(tǒng)計學意義(P0.01)。(6)用5LD50攻擊小鼠,各組21天生存率分別為:pcDNA3/MDC-L-15-VP1組和pcDNA3/MDC-L19-VP1組均為37.5%,pcDNA3/MDC-L10- VP1組25.0%,pcDNA3/VP1組12.5%, pcDNA3組11天內全部死亡。(7)隨Linker長度的增加血中病毒滴度依次降低, pcDNA3/MDC-L19-VP1組小鼠和其它各組相比差別均有統(tǒng)計學意義。(8)心肌病理學檢查隨Linker長度的增加心肌病理改變依次減輕,但并無明顯異。 結論:(1)用PCR方法成功擴增MDC-L15、MDC-L19、L15-VP1和L19-VP1基因片段。(2)成功構建真核表達質粒pcDNA3/MDC-L15-VP1、pcDNA3/MDC-L19-VP1。(3)用5LD50的CVB3攻擊后,小鼠的生存率隨著Linker長度的加長有一定的提高。(4)小鼠血清中和抗體滴度隨免疫次數(shù)的增加而提高,第三次免疫后pcDNA3/MDC-L19-VP1組中和抗體滴度最高,與其它各組相比差異均有統(tǒng)計學意義。(5)特異性淋巴細胞增殖實驗和CTL殺傷實驗結果表明pcDNA3/MDC-L19-VP1組增殖指數(shù)和殺傷率最高,與其它各組相比差異均有統(tǒng)計學意義。(6)中和抗體滴度、細胞免疫檢測、血中病毒滴度、和心肌切片病理學分析結果一致,說明融合基因疫苗pcDNA3/MDC-L19-VP1是較理想的一種疫苗。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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