發(fā)布時(shí)間：2018-07-28 20:08

【摘要】： 呼吸道合胞病毒(Respiratory syncytial virus，RSV)是世界范圍內(nèi)引起嬰幼兒下呼吸道感染的主要病原體之一，全球每年有近四百萬(wàn)兒童受其感染，給人們?cè)斐闪司薮蟮呢?cái)產(chǎn)損失和精神痛苦，目前還沒(méi)有一種安全有效的疫苗問(wèn)世。本研究首次通過(guò)替換G蛋白的CX3C模序來(lái)降低肺部嗜酸性粒細(xì)胞的數(shù)目，通過(guò)改造過(guò)的G蛋白與粘膜佐劑LT物理混合后免疫小鼠，增強(qiáng)疫苗在呼吸道的sIgA，來(lái)增強(qiáng)疫苗免疫的效果。 人工合成G蛋白基因的部分核酸序列，并通過(guò)PCR將其中所存在的CX3C模序替換為RSV M蛋白上的CTL表位，構(gòu)建表達(dá)載體G／pET22b和改造過(guò)的G(CTL)／pET22b，轉(zhuǎn)入大腸桿菌BL21(DE3)中，，G蛋白和G(CTL)蛋白在大腸桿菌中均有高效表達(dá)。在37℃下誘導(dǎo)的蛋白占總蛋白的30％以上。應(yīng)用His-Tag與金屬離子的親和力對(duì)目的蛋白進(jìn)行了純化，純度大于92％。 用純化的目的蛋白與無(wú)毒型菌熱不穩(wěn)定腸毒素(LT)進(jìn)行物理混合后進(jìn)行免疫，分別設(shè)立A：PBS；B：LT；C：G(CTL)蛋白+LT；D：G蛋白(含CX3C)。共四組，分別在0、1、4周進(jìn)行免疫。免疫后，針對(duì)血清中IgG、IgA和呼吸道上的sIgA及嗜酸性粒細(xì)胞的數(shù)量進(jìn)行測(cè)定。結(jié)果表明：C組和D組小鼠血清中IgG明顯高于A組和B組，幾何平均滴度分別為1995.26和1584.89。IgA只出現(xiàn)在C組的血清和呼吸道中，幾何平均滴度分別為1781.23和3.167，而在A、B、D組都沒(méi)有；C組小鼠血清和呼吸道內(nèi)的嗜酸性粒細(xì)胞的數(shù)目與A組沒(méi)有顯著性的差異，但D組的嗜酸性粒細(xì)胞數(shù)目明顯高于其它兩組。 本研究成功地表達(dá)了RSV G蛋白和G(CTL)蛋白，對(duì)其進(jìn)行純化，并對(duì)疫苗樣品的免疫原性進(jìn)行測(cè)定。針對(duì)G蛋白CX3C模序的改造使肺內(nèi)嗜酸性粒細(xì)胞的數(shù)量相比對(duì)照組沒(méi)有增加。通過(guò)G蛋白與粘膜佐劑LT混合后免疫小鼠，不僅提高了小鼠血清中的抗體滴度，而且還使血清和呼吸道中IgA的分泌增加，增強(qiáng)疫苗的粘膜免疫效果。呼吸道合胞病毒G蛋白亞單位粘膜疫苗的成功構(gòu)建，將為RSV疫苗的開(kāi)發(fā)開(kāi)辟一條新的途徑，為進(jìn)一步研究G蛋白在RSV的致病機(jī)理中的作用和研制RSV疫苗奠定基礎(chǔ)。
[Abstract]:Respiratory syncytial virus (RSV) is one of the main pathogens of infantile lower respiratory tract infection in the world. Nearly 4 million children worldwide are infected with RSV every year, which has caused great property loss and mental suffering to people. There is no safe and effective vaccine yet. In this study, the CX3C motif of G protein was replaced to reduce the number of eosinophil in lung for the first time. The immunized mice were immunized with modified G protein and mucosal adjuvant LT to enhance the immune effect of the vaccine in the respiratory tract. Some nucleic acid sequences of G protein gene were synthesized, and the existing CX3C motifs were replaced by CTL epitopes on RSV M protein by PCR. The expression vector G/pET22b and modified G (CTL) / pET22b were constructed and transferred into Escherichia coli BL21 (DE3). Both G protein and G (CTL) protein were highly expressed in E. coli. The protein induced at 37 鈩

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