微生物學(xué)通報(bào) 20 June 2009, 36(6):936～942
肺炎球菌表面蛋白A(PspA)的克隆表達(dá)及交叉免疫作用
林子琳1 林海英1 汪媛媛1 白鍇凱1 郭養(yǎng)浩1,2*
(1. 福州大學(xué)藥物生物技術(shù)研究所 福建 福州 350002)
(2. 福建省醫(yī)療儀器和醫(yī)藥技術(shù)重點(diǎn)實(shí)驗(yàn)室 福建 福州 350002)
摘 要: 本工作從中國(guó)臨床最常見(jiàn)的5種莢膜血清型肺炎鏈球菌(FY01, FY05, FY6B, FY19F, FY23F)克隆獲得編碼PspA蛋白N端α-螺旋區(qū)的基因片段。分別將5種PspA基因片段克隆到表達(dá)載體pET-27b(+)上, 成功構(gòu)建重組質(zhì)粒pET-pspA, 轉(zhuǎn)化大腸桿菌BL21(DE3),。經(jīng)乳糖誘導(dǎo)表達(dá)和親和層析分離, 獲得高純度重組蛋白,。通過(guò)家族專(zhuān)屬引物PCR的方法鑒定這5種莢膜血清型肺炎球菌的PspA蛋白所屬家族, 并進(jìn)一步根據(jù)基因測(cè)序結(jié)果通過(guò)生物信息學(xué)方法鑒定了所屬支系。FY01 rPspA,、FY6B rPspA,、FY19F rPspA同屬于家族Ⅰ支系1, FY05 rPspA屬于家族Ⅰ支系2, FY23F rPspA屬于家族Ⅱ支系3。以FY01 rPspA免疫小鼠, 在20 μg/mL免疫劑量時(shí)抗體滴度達(dá)到1: 210000, 顯示了重組蛋白較好的免疫原性,。Western blotting交叉免疫反應(yīng)性研究表明, 抗FY01 rPspA抗血清與FY01 rPspA,、FY6B rPspA和FY19F rPspA反應(yīng)性較好, 而與另外兩種重組蛋白幾乎無(wú)反應(yīng), 提示PspA交叉免疫作用僅限于本支系內(nèi)。動(dòng)物保護(hù)實(shí)驗(yàn)表明, FY01 rPspA免疫的小鼠對(duì)FY6B,、FY01 兩種菌的攻擊具有較好的保護(hù)作用, 對(duì)FY05,、FY23F的攻擊未見(jiàn)明顯的保護(hù)作用。本研究成果對(duì)于研制高效肺炎球菌疫苗具有重要的指導(dǎo)意義,。
關(guān)鍵詞: 肺炎球菌表面蛋白A(PspA), 交叉保護(hù)作用, 家族, 支系, 克隆表達(dá)
Cloning and Expression of Pneumococcal Surface Protein A and Its Cross-immunoreactivity
LIN Zi-Lin1 LIN Hai-Ying1 WANG Yuan-Yuan1 BAI Kai-Kai1 GUO Yang-Hao1,2*
(1. Institute of Pharmaceutical Biotechnology and Engineering, College of Biological Science and Biotechnology, Fuzhou, Fujian 350002, China)
(2. Fujian Key Laboratory of Medical Instrumentation and Pharmaceutical Technology, Fuzhou, Fujian 350002, China)
Abstract: In this study, we cloned genes coding the N-terminal region of PspA( pneumococcal surface pro-tein A) from five of the most epidemic pneumoniae serotypes in China(FY01, FY05, FY6B, FY19F, FY23F). The obtained genes were respectively constructed into prokaryotic expression vector pET-27b(+), and then recombinant plasmids pET-pspA were respectively transformed into Escherichia coli BL21(DE3) for PspA expression. Induced with lactose, the five recombinant proteins were highly expressed and then purified by Ni-chelation chromatography. PCR with Family special primers was carried out to identify Family of PspA, and Clade of PspA was identified by means of bioinformatics according to the sequencing results.FY01rPspA, FY6B rPspA and FY19FrPspA belonged to Clade 1 of FamilyⅠ, FY05rPspA belonged toClade 2 from FamilyⅠand FY23F rPspA belonged to Clade 3 from FamilyⅡ. Strong immunogenicity had been demonstrated in mice immuned with FY01rPspA at dose of 20 μg/mL each, of which antiserum anti-body titer reached 1:210000. Result of western blotting showed that FY01rPspA antiserm reacted well with Y6B rPspAand FY19FrPspA but poorly with the other two, which suggested that cross-immunoreactivity was restricted in the same Clade. Mice immuned with FY01rPspA protected well against FY6B, while against FY05, FY23F was not effectively protected. The result in this study has much instructive signifi-cance for the development of a new efficient vaccine against pneumoniae.
Keywords: Pneumococci surface protein A(PspA), Clone and expression, Cross-immunoprotection, Family of PspA, Clade of PspA
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