【摘要】：呼吸道合胞病毒(Respiratory syncytial virus, RSV)是一種能導(dǎo)致嬰幼兒下呼吸道感染的主要病原體。感染后出現(xiàn)急性支氣管炎、肺炎、嚴重者甚至死亡。截止目前還沒有一種安全有效的疫苗得到應(yīng)用。本研究通過將截短的RSV G 130-230位氨基酸(包含有CX3C模序)與GCTL(用CTL表位替換了CX3C)或輔以黏膜佐劑后免疫Balb/c小鼠,來分析和評價疫苗對小鼠免疫功能的影響。 Gcx3c蛋白和GCTL蛋白在大腸桿菌中表達。表達的蛋白,應(yīng)用His-Tag與Co2+的親和力對蛋白進行純化。將純化后的Gcx3c蛋白、GCTL蛋白分別與無毒型大腸桿菌不耐熱腸毒素(Heat-labil enterotoxin, LT)混合后免疫Balb/c小鼠,設(shè)立Gcx3c組；GCTL組；GCTL組；GCTL+LT組；LT組；PBS組,共6組,分別在0,1,4周免疫。免疫結(jié)束后,針對血清中的IgG、IgA、IgG2a/IgG1、呼吸道sIgA、組胺、嗜酸性粒細胞以及淋巴細胞體外增殖、CTL應(yīng)答效應(yīng)等免疫指標分別通過ELISA、HE染色以及MTT染色法等進行測定。用107 pfu/ml RSV滴鼻攻擊免疫后的小鼠,連續(xù)3天。攻毒結(jié)束后處死小鼠,計數(shù)嗜酸性粒細胞,檢測組胺水平,并制備組織切片。結(jié)果使用SPSS16.0軟件進行分析。 Gcx3c蛋白和GCTL蛋白在大腸桿菌中以包涵體的形式大量表達,在37℃下誘導(dǎo)的包涵體占到總蛋白的40%以上。應(yīng)用His-tag與金屬離子的親和力純化后純度達到95%以上,Western blotting結(jié)果證明所表達的是目的蛋白。 免疫檢測結(jié)果表明：血清IgG在第二次免疫后,GCTL、GCTL+LT組抗體滴度顯著高于Gcx3c+LT及Gcx3c(P0.05),且以上四組均高于LT及PBS組(P0.001)：血清IgA在第二次免疫后,GCTL、GCTL+LT組抗體滴度顯著高于Gcx3c及Gcx3c+LT(P0.05),且以上四組均高于LT及PBS組(P0.05)；而在sIgA中,加了LT佐劑的兩個實驗組的sIgA抗體滴度顯著高于Gcx3c、GCTL、LT、PBS組(P0.05)；血清IgG2a/IgG1中GCTL組平衡效果優(yōu)于GCTL+LT、GCX3C+LT、GCX3C組(P0.05),GCTL組在用CTL表位替換了CX3C模序后,Th1/Th2趨于平衡,并且還發(fā)現(xiàn)加入了佐劑LT后能發(fā)揮一個協(xié)調(diào)Th1/Th2的生理作用,有調(diào)節(jié)免疫反應(yīng)極化的情況；淋巴細胞體外增殖實驗中,抗原刺激后,只有相應(yīng)的Gcx3c和GCTL蛋白組細胞出現(xiàn)了增殖,對照組和佐劑組都沒有增殖,且蛋白組細胞增殖量顯著高于對照組(P0.05)；比較Gcx3C、GcTL組以及PBS組的組胺,GCX3C組胺含量明顯高于其他兩組(P0.05),GcTL與PBS組間無顯著差異(P0.05)；Gcx3c+LT組的嗜酸性粒細胞數(shù)量高于GCTL、GCTL+LT、PBS組(P0.05),而后三組之間數(shù)量無顯著差異(P0.05)；組織切片結(jié)果也顯示,GCTL蛋白組小鼠肺部沒有出現(xiàn)炎癥反應(yīng),而GCX3C蛋白組則有明顯的炎癥反應(yīng)；RSV特異性CTL應(yīng)答結(jié)果表明,GCTL組誘導(dǎo)了顯著的RSV特異性CTL殺傷活性,而GCX3C在針對病毒時也存在一定程度的殺傷。 攻毒檢測結(jié)果表明：攻毒后GCTL組嗜酸性粒細胞數(shù)量顯著低于GCX3C組、PBS組和LT組(P0.05)；GCX3C組的組胺含量顯著高于GCTL組(P0.05)；肺表和組織切片結(jié)果顯示,GCTL蛋白組小鼠肺沒有明顯病變,而GCX3C組、PBS組和LT組則出現(xiàn)了嚴重的病變。 本實驗成功的表達純化了GCX3C、GCTL和LT蛋白,并就疫苗對小鼠免疫功能的影響和攻毒后的保護能力進行了測定。實驗結(jié)果顯示,混以LT佐劑后,提高了黏膜部的sIgA的量。針對GCX3C進行的改造是成功有效的,GCTL增強了對RSV特異性的CTL應(yīng)答反應(yīng),并且有效的降低了呼吸道組胺和嗜酸性粒細胞的數(shù)量,避免了再次感染的過激反應(yīng)。攻毒后的部分實驗結(jié)果也從一個側(cè)面體現(xiàn)了GCTL在保護性方面發(fā)揮了應(yīng)有的作用。呼吸道合胞病毒G蛋白亞單位疫苗的小鼠實驗,為進一步的研究RSV致病機理及RSV疫苗的研發(fā)奠定了基礎(chǔ)。
[Abstract]:Respiratory syncytial virus (RSV) is a major pathogen that can lead to lower respiratory tract infection in infants. Acute bronchitis, pneumonia, and even death after infection. The application of a safe and effective vaccine is not yet available. The effects of the vaccine on the immune function of the mice were analyzed and evaluated by comparing the truncated RSV G 130-230 amino acids (including the CX3C mold sequence) with the GCTL (replacing the CX3C with the CTL epitope) or by immunizing the Balb/ c mice with a mucosal adjuvant. Gcx3c protein and GCTL protein in Escherichia coli D. expressed protein, and the affinity of His-Tag and Co2 + is used for the purification of the protein. The purified Gcx3c protein and the GCTL protein were mixed with the non-toxic E. coli heat-labile enterotoxin (LT) to immunize the Balb/ c mice to set up the Gcx3c group, the GCTL group, the GCTL group, the GCTL + LT group, the LT group, the PBS group, the total 6 groups, respectively, at 0,1 and 4 weeks. The immune indexes such as IgG, IgA, IgG2a/ IgG1, sIgA, histamine, eosinophils and lymphocytes in the serum were measured by ELISA, HE staining and MTT staining. Determination. Mice immunized with 107 pfu/ ml RSV nasal attack, continuous 3 d. The mice were sacrificed after the challenge, the eosinophils were counted, the histamine level was detected, and the tissue was prepared The results are separated using the SPSS16.0 software The Gcx3c protein and the GCTL protein were expressed in the form of inclusion bodies in E. coli, and the inclusion bodies induced at 37 鈩，

本文編號：2501057