發(fā)布時間：2018-06-19 18:56

  本文選題：VP1 + 柯薩奇病毒B組3型(CVB3)��； 參考：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:柯薩奇病毒屬于小RNA病毒科腸道病毒屬。其中B組3型(Coxsackievirus group B type 3, CVB3)所致的病毒性心肌炎嚴(yán)重危害人類健康,也是兒童和青少年猝死的重要原因,迄今尚無有效的預(yù)防手段。VP1是CVB3主要的衣殼蛋白,含有多個T、B細(xì)胞表位,可誘導(dǎo)機(jī)體產(chǎn)生免疫應(yīng)答。 基因工程亞單位疫苗是將目的基因克隆到原核或真核表達(dá)載體中,在原核或真核細(xì)胞中表達(dá),經(jīng)純化制備的疫苗。亞單位疫苗具有較強(qiáng)的免疫原性,成分單一,可顯著提高抗體水平,免除非抗原成分引起的副作用,保證疫苗的安全性。本室前期將VP1基因克隆到原核表達(dá)載體,在大腸桿菌成功表達(dá)了VP1蛋白,經(jīng)純化后免疫小鼠。結(jié)果顯示VP1蛋白能誘導(dǎo)產(chǎn)生較高水平的體液免疫和細(xì)胞免疫應(yīng)答,但未能達(dá)到滿意的保護(hù)效果。因此,進(jìn)一步增強(qiáng)VP1蛋白的免疫原性,提高CVB3特異性體液和細(xì)胞免疫應(yīng)答,已成為倍受關(guān)注的研究方向。 有多種因素影響機(jī)體對抗原的免疫應(yīng)答強(qiáng)度及類型,主要是抗原物質(zhì)本身的性質(zhì)和進(jìn)入機(jī)體的途徑、劑量、次數(shù)、免疫間隔時間以及免疫佐劑的類型等。本研究用原核細(xì)胞表達(dá)得到純化CVB3VP1蛋白,選取3種常用免疫途徑,3種免疫劑量,聯(lián)合3種免疫佐劑共同免疫小鼠。觀察不同免疫途徑,不同劑量和不同佐劑對免疫效果的影響。 方法: 1將我室構(gòu)建的原核表達(dá)質(zhì)粒pET-his/VP1轉(zhuǎn)入表達(dá)宿主菌BL21(DE3)pLysS,誘導(dǎo)表達(dá)攜帶有6×HIS標(biāo)簽的VP1蛋白,經(jīng)SDS-PAGE和Western-blot鑒定。 2將大量誘導(dǎo)表達(dá)后的菌體在冰浴中超聲破碎,分離上清和包涵體沉淀,經(jīng)SDS-PAGE分析,結(jié)果表明蛋白以包涵體形式存在,用尿素溶解沉淀,利用金屬鎳離子親和層析柱純化目的蛋白,透析后濃縮蛋白。 3動物實驗:分二期進(jìn)行。第一期將小鼠隨機(jī)分為3組,每組12只,分別為腹腔注射組、皮下注射組、肌肉注射組。純化后的蛋白以PBS稀釋后,經(jīng)過腹腔、頸背部皮下和股四頭肌注射免疫小鼠。初次免疫加完全弗氏佐劑,以后免疫用不完全弗氏佐劑。每次每只接種100μg;每3周免疫1次,共免疫3次;每次免疫后第14天,內(nèi)眥靜脈取血,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特異性中和抗體的水平;用純化VP1抗原包被酶聯(lián)板,采用ELISA方法檢測小鼠血清抗體IgG抗體效價。經(jīng)過比較選擇最佳免疫注射途徑,進(jìn)行第二期動物實驗。 4第二期動物實驗:將6～8周齡雄性BALB/c小鼠隨機(jī)分為6組,每組18只。純化后的蛋白以PBS稀釋后,采用肌肉注射途徑免疫,①低劑量組:每次每只接種50μg。②中劑量組:每次每只接種100μg。③高劑量組:每次每只接種150μg。以上3組初次免疫加完全弗氏佐劑,以后免疫用不完全弗氏佐劑。④氫氧化鋁佐劑組:每次每只接種100μg。⑤ISA 720組:每次每只接種100μg。⑥PBS組。每3周免疫1次,共免疫3次;每次免疫后第14天,內(nèi)眥靜脈取血,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特異性中和抗體的水平;用純化VP1抗原包被酶聯(lián)板,采用ELISA方法檢測小鼠血清特異性抗體效價。第3次免疫后3周,每組3只小鼠取脾臟制備淋巴細(xì)胞懸液,采用細(xì)胞計數(shù)試劑盒(cell counting kit-8, CCK-8)法進(jìn)行特異性淋巴細(xì)胞增殖活性和特異性細(xì)胞毒性T淋巴細(xì)胞(cytotoxic T lymphocyte, CTL)殺傷活性的檢測;另每組12只小鼠用致死量的CVB3(4LD_(50))進(jìn)行腹腔注射,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CVB3(3LD_(50))攻擊,注射病毒后第7天處死,用于血中病毒滴度的測定。 結(jié)果: 1 SDS-PAGE和Western-blot證實原核表達(dá)質(zhì)粒pET-his/VP1轉(zhuǎn)化的大腸桿菌BL21(DE3)pLysS成功表達(dá)了CVB3 VP1蛋白,蛋白主要以包涵體形式存在。 2不同免疫途徑各組各次免疫后血清中和抗體滴度分別為:腹腔注射組: 1:7.08,1:28.18,1:42.66;皮下注射組:1:7.08,1:11.22,1:35.48;肌肉注射組:1: 8.91,1:47.86,1:70.79。血清特異性IgG抗體分別為:皮下注射組:1:155,1: 7600,1:11800;腹腔注射組1:240, 1:8300,1:17000;肌肉注射組1:425,1:22400,1:38400。單因素方差分析表明,三次免疫后,各組中和抗體和特異性IgG抗體滴度逐次增加(P0.05)。三次免疫后,中和抗體和特異性IgG抗體:肌肉注射組腹腔注射組皮下注射組(P0.01)。 3經(jīng)肌肉注射途徑免疫后,不同劑量各組血清中和抗體效價分別為:低劑量組:1:6.31, 1:42.66, 1:47.86;中劑量組:1:7.08, 1:44.67, 1:63.10;高劑量組: 1:7.08, 1:50.12,1:70.79。血清特異性IgG水平:低劑量組: 1:200, 1:6400, 1: 19200;中劑量組: 1:398, 1:21000, 1:36300;高劑量組: 1:425, 1:23040, 1:51200。單因素方差分析表明,三次免疫后,各組中和抗體和特異性IgG抗體滴度逐次增加(P0.05)。末次免疫后各組比較差別有統(tǒng)計學(xué)意義。中和抗體和特異性IgG抗體:高劑量組中劑量組低劑量組(P0.05)。 4不同佐劑組各次免疫后血清中和抗體滴度為:氫氧化鋁佐劑組: 1:5.62, 1:12.59, 1:26.92;弗氏佐劑(中劑量)組: 1:7.08, 1:44.67, 1:63.10; ISA720佐劑組: 1:8.91, 1: 44.67, 1: 85.11。各次免疫后血清特異性IgG水平:氫氧化鋁佐劑組: 1:150, 1:1600,1:11800;弗氏佐劑(中劑量)組:1:398, 1:21000, 1:36300; ISA720佐劑組:1:280, 1:12800,1:38400。單因素方差分析表明,三次免疫后,各組中和抗體和特異性IgG抗體滴度逐次增加(P0.05)。末次免疫后各組比較差別有統(tǒng)計學(xué)意義。中和抗體:ISA720佐劑組弗氏佐劑組氫氧化鋁佐劑組(P0.05)。特異性IgG抗體:ISA720組和弗氏佐劑組高于氫氧化鋁佐劑組,(P0.05)弗氏佐劑組和ISA720組差別無統(tǒng)計學(xué)意義(P0.05)。 5小鼠脾臟淋巴細(xì)胞增殖活性,以CVB3或Con A刺激,各組均高于PBS組(P0.05);以CVB3刺激,高劑量組高于低劑量組和中劑量組(P0.05); ISA720佐劑組高于弗氏佐劑組和氫氧化鋁佐劑組(P0.05)。ConA刺激時,各組均顯著高于PBS組(P0.05)。各組之間差別無統(tǒng)計學(xué)意義。 6小鼠脾臟特異性CTL殺傷活性,各組均顯著高于PBS組(P0.05);高劑量組高于中劑量組和低劑量組(P0.05);弗氏佐劑組和ISA720組高于氫氧化鋁組(P0.05)。 7用4LD_(50)攻擊小鼠,PBS組、低劑量組、中劑量組、高劑量組、氫氧化鋁佐劑組、ISA720佐劑組的21天生存率分別為: 0、66.67%、66.67%、50%、33.33%和50 %。經(jīng)χ2檢驗分析,各組之間無明顯差異(P0.05)。經(jīng)Kaplan-Meier法進(jìn)行生存分析表明,各組生存率曲線分布有差別(P0.05)。低劑量組和中劑量組的生存時間好于高劑量組,弗氏佐劑組生存時間好于氫氧化鋁佐劑組(P0.05)。 8各組血清病毒滴度均比PBS組低(P0.05)。單因素方差分析顯示,高劑量組血中病毒滴度低于低、中劑量組(P0.05);ISA720佐劑組低于氫氧化鋁組(P0.05)。 結(jié)論: 1成功在原核細(xì)胞表達(dá)了CVB3 VP1蛋白,并進(jìn)行了純化。 2三種免疫途徑接種,各組血清中和抗體和IgG抗體滴度均隨免疫次數(shù)的增加而提高,肌肉免疫能誘導(dǎo)更高的體液免疫水平。 3采用肌肉注射途徑免疫,中和抗體和IgG抗體水平隨免疫劑量增加而提高。用致死量病毒攻擊后,病毒載量隨免疫劑量增加而降低,但低劑量組的生存時間好于其他劑量組。 4三種佐劑比較,弗氏佐劑和ISA 720佐劑的對VP1蛋白的免疫增強(qiáng)作用優(yōu)于氫氧化鋁佐劑。但是弗氏佐劑不能應(yīng)用于人,因此ISA 720佐劑是一種值得進(jìn)一步研究的有效佐劑。 5采用合適劑量(50-100μg /只)的VP1蛋白,應(yīng)用弗氏佐劑或ISA 720佐劑,選擇肌肉注射途徑免疫可獲得較好的免疫效果。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 岳艷;徐薇;蔣正剛;胡林昆;李康;熊思東;;C家族趨化因子XCL1增強(qiáng)CVB3基因疫苗的抗病毒免疫應(yīng)答及保護(hù)作用[J];現(xiàn)代免疫學(xué);2008年02期

2 劉純杰;疫苗研制技術(shù)的現(xiàn)狀與展望[J];微生物學(xué)免疫學(xué)進(jìn)展;2002年03期

3 房文亮,王永祥,金玉懷,金士香,于丹軍,方艷輝;CVB3 VP1和hIL-15基因真核雙表達(dá)質(zhì)粒的構(gòu)建及免疫效果觀察[J];中華微生物學(xué)和免疫學(xué)雜志;2003年03期

4 韓小艷;趙娜;高志云;揣俠;羨鮮;藍(lán)佳明;張永紅;王永祥;;接頭長度對MDC與CVB3VP1融合基因疫苗免疫效果的影響[J];中華微生物學(xué)和免疫學(xué)雜志;2007年12期

5 趙娜;韓小艷;王曉凌;李劍;李偉;謝立新;李琳;王永祥;;C3d對分泌型柯薩奇病毒B組3型VP1 DNA疫苗的免疫增強(qiáng)作用[J];中華醫(yī)學(xué)雜志;2007年36期

6 李偉;藍(lán)佳明;李劍;金玉懷;高志云;揣俠;劉貴霞;張永紅;王永祥;;柯薩奇病毒B組3型VP1蛋白的原核表達(dá)及免疫效果研究[J];中國免疫學(xué)雜志;2009年04期

7 張?zhí)煲?王華;張春輝;朱昌來;林琳;顧君一;;gp96-肽復(fù)合物體外誘導(dǎo)CTL反應(yīng)及其抗腫瘤效應(yīng)研究[J];腫瘤;2005年06期

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