本文選題：PRRSV + GP5��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2010年碩士論文

【摘要】：【目的】為研究LTB的佐劑特性,在對(duì)LTB和GP5進(jìn)行了表達(dá)條件及純化方法優(yōu)化的基礎(chǔ)上,以LTB為免疫佐劑,GP5為模式抗原進(jìn)行小鼠及仔豬免疫試驗(yàn),為研究新型PRRSV疫苗探尋新途徑。 【方法】對(duì)GP5蛋白原核表達(dá)的誘導(dǎo)時(shí)間、IPTG濃度、表達(dá)菌裂解液及表達(dá)蛋白純化條件進(jìn)行了優(yōu)化;對(duì)LTB蛋白真核表達(dá)的誘導(dǎo)時(shí)間、甲醇濃度、表達(dá)培養(yǎng)基蛋白濃度、表達(dá)菌起始濃度及表達(dá)蛋白純化方法進(jìn)行了優(yōu)化;將雌性ICR小鼠分為8組,每組10只,分別為PBS陰性對(duì)照組、GP5+弗氏佐劑陽(yáng)性對(duì)照組、GP5肌肉注射組、GP5+LTB肌肉注射組、GP5皮下注射組、GP5+LTB皮下注射組、GP5滴鼻組和GP5+LTB滴鼻組,三免后以間接ELISA法檢測(cè)血清抗體,以MTT法檢測(cè)淋巴細(xì)胞增殖;將1～2日齡仔豬隨機(jī)分為4組,每組10只,分別為GP5滴鼻組、GP5+LTB滴鼻組、GP5注射組及GP5注射+LTB滴鼻組,二免后以間接ELISA法檢測(cè)血清抗體。 【結(jié)果】GP5蛋白表達(dá)及純化最佳條件:1mmol/L的IPTG誘導(dǎo)3.5h,裂解液為pH7.8的8mol/L尿素,洗滌液為pH5.0和pH4.5的8mol/L尿素,洗脫液為pH4.0的8mol/L尿素,表達(dá)蛋白能與PRRSV陽(yáng)性豬血清發(fā)生特異性反應(yīng),而與陰性血清不反應(yīng)。LTB蛋白表達(dá)及純化最佳條件:在1×BMMY中培養(yǎng)表達(dá)菌至OD600=4,1%(W/W)甲醇誘導(dǎo)96h,55%飽和硫酸銨沉淀后Ni-NTA柱一次親和層析,D(+)-Immobilized galactose二次親和層析,純化后的LTB以五聚體形式存在,具有結(jié)合GM1的生物學(xué)活性;小鼠三免后GP5+弗氏佐劑組血清抗體水平最高,GP5+LTB免疫組抗體水平顯著高于GP5免疫組,肌肉注射免疫組抗體水平明顯高于皮下注射組,滴鼻免疫組沒(méi)有檢測(cè)到特異性血清抗體;MTT法檢測(cè)結(jié)果表明,GP5+弗氏佐劑組、GP5+LTB肌肉注射和GP5+LTB皮下注射組B淋巴細(xì)胞增殖顯著,除了GP5+弗氏佐劑組外,其他各免疫組均無(wú)明顯的脾臟T淋巴細(xì)胞增殖反應(yīng)。仔豬實(shí)驗(yàn)表明,LTB具有一定的黏膜免疫增強(qiáng)作用,GP5+LTB免疫組抗體水平高于GP5免疫組,GP5注射+LTB滴鼻免疫組血清抗體水平增幅大于GP5+LTB滴鼻組。 【結(jié)論】建立了GP5原核表達(dá)純化及LTB真核表達(dá)純化的最優(yōu)方法;小鼠免疫試驗(yàn)表明,LTB經(jīng)肌肉和皮下途徑免疫后能夠有效地增強(qiáng)GP5蛋白的免疫原性,具有良好的體液免疫增強(qiáng)作用;仔豬免疫試驗(yàn)表明,LTB通過(guò)滴鼻方式免疫同樣可以增強(qiáng)GP5蛋白的免疫原性,具有一定的黏膜佐劑作用。
[Abstract]:[objective] in order to study the adjuvant characteristics of LTB, on the basis of optimizing the expression conditions and purification methods of LTB and GP5, the mice and piglets were immunized with LTB as model antigen, and a new way to study new PRRSV vaccine was explored. [methods] the induction time of prokaryotic expression of GP5 protein, the concentration of IPTG, the lytic solution of expression bacteria and the purification conditions of LTB protein were optimized, and the induction time, methanol concentration and protein concentration of expression medium of LTB protein were optimized. The initial concentration of expression bacteria and the purification method of expressed protein were optimized, and female ICR mice were divided into 8 groups with 10 mice in each group. The PBS negative control group with Freund's adjuvant positive control group with GP5 intramuscular injection group with GP5 LTB intramuscular injection group and the subcutaneous injection group with GP5 LTB subcutaneous injection group with GP5 nose dropping and GP5 LTB nose dropping group were used to detect the serum antibody by indirect ELISA method after three immunizations. Lymphocyte proliferation was detected by MTT, and the piglets were randomly divided into 4 groups, 10 in each group: GP5 nose dropping group (GP5 group), GP5 LTB nasal drip group (GP5 group) and GP5 injection group (LTB nasal drip group). Serum antibodies were detected by indirect ELISA method after two immunizations. [results] the optimal conditions for the expression and purification of GP5 protein were as follows: 1 mmol / L IPTG was induced 3.5 h, the lysate was 8mol/L urea of pH7.8, the washing solution was 8mol/L urea of pH5.0 and pH4.5, and the eluate was 8mol/L urea of pH4.0. The expressed protein could react specifically with PRRSV positive pig serum. However, the optimal conditions for expression and purification of unreacted. LTB protein with negative serum were as follows: the expressed bacteria were cultured in 1 脳 BMMY to OD600 / 4L / W) methanol was induced to precipitate with 55% saturated ammonium sulfate at 96 h, and then Ni-NTA column one-step affinity chromatography (Ni-NTA) -immobilized galactose secondary affinity chromatography was used. The purified LTB existed in the form of pentamer. The antibody level of GP5 Freund's adjuvant group was significantly higher than that of GP5 immunization group, and the antibody level of GP5 Freund's adjuvant group was significantly higher than that of subcutaneous injection group, while the antibody level of GP5 LTB immunized group was significantly higher than that of GP5 immunized group. No specific serum antibody was detected in nasal drip immunization group. The results showed that the proliferation of B lymphocytes was significant in GP5 LTB intramuscular injection group and GP5 LTB subcutaneous injection group, except for GP5 Freund's adjuvant group. There was no obvious proliferation of spleen T lymphocytes in other immune groups. The results showed that LTB had a certain enhancement effect on mucosal immunity. The antibody level of GP5 LTB immunized group was higher than that of GP5 immunization group. The increase of serum antibody level in LTB nasal immunization group was higher than that in GP5 LTB nasal drip group. [conclusion] the optimal method of GP5 prokaryotic expression purification and LTB eukaryotic expression purification was established, and mouse immunological test showed that the immunogenicity of GP5 protein could be effectively enhanced by muscle and subcutaneous immunization. The immune test of piglets showed that LTB could also enhance the immunogenicity of GP5 protein by nasal drip and had a certain mucosal adjuvant effect.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

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1 田文標(biāo),鄒全明,吳超,張衛(wèi)軍,楊s，

本文編號(hào)：1933635