北京地区两株人偏肺病毒M蛋白基因的原核表达及抗原活性分析

Human metapneumovirus(hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms.Matrix protein(M) is one of the most important structural proteins.For further studying of hMPV,the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCm-M1817 were cloned by PCR and sub-cloned into the pET30a(+) vector,which is a prokaryotic expression vector,after dual-enzyme digestion with Bam HI and Xho I.The positive recombinated plasmids were transformed into E.coli BL21(DE3) and expressed under the inducing of IPTG.Target proteins were characterized by SDSPAGE and Western blotting.In this article,we’ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6·His-N were highly produced after inducing by 1mmol/L IPTG at 37℃.A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE.Most of the target protein existed in inclusion body.Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV.So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities.It can be used for further studying of hMPV infections in Beijing.

北京地区人偏肺病毒核蛋白基因的原核表达及抗原活性分析

目的用大肠杆菌表达获得人偏肺病毒主要结构蛋白N蛋白,为下一步的深入研究奠定基础。方法从重组质粒pUCm-N1816中PCR扩增获得N基因,用BamHⅠ和EcoRⅠ双酶切后插入到原核表达载体pET30a(+)中,得到重组表达质粒pET30a-N1816,转化大肠杆菌BL21(DE3),IPTG诱导培养。SDS-PAGE和Westernblot检测目的蛋白的表达和抗原性。结果经双酶切和测序证明1.2kbN1816基因正确插入pET30a中,并具有正确的读码框架,得到预期的pET30a-N1816重组原核表达质粒。37℃,1mmol/LIPTG诱导培养6h后产生大量带6个组氨酸标记的重组N蛋白,主要以包涵体形式存在,占细胞总蛋白的20%左右。N蛋白粗提物经Co2+亲和层析获得较理想纯化。Westernblot结果显示,体外所表达的蛋白能和特异性抗血清及人血清反应。结论本研究成功构建了重组pET30a-N1816原核表达质粒,N蛋白获得了高效表达,并且具有特异抗原活性,可用于人偏肺病毒的深入研究。

恙虫病东方体合肥分离株截短56kDa蛋白抗原表达及活性分析

本文获得恙虫病东方体合肥分离株截短56kDa外膜蛋白基因工程纯化抗原，并鉴定其免疫学活性，为进一步研制广谱诊断试剂、疫苗以及分析其在东方体致病的分子机制奠定基础。我们通过DNA Star和Mega等软件对含有440个氨基酸的合肥株56kDa蛋白序列进行抗原性分析；将合肥株56kDa截短蛋白基因克隆至原核表达载体pET28a获重组质粒，转入大肠杆菌E．coli BL21感受态中，经IPTG诱导，SDS—PAGE电泳分析检测蛋白表达；采用亲和层析法纯化目的蛋白，Western-blot及ELISA法鉴定其免疫学活性。序列分析发现合肥分离株56kDa蛋白可分为4个保守区和3个可变区，抗原性分析结果显示，其保守区的亲水性和抗原性均有很高峰值。SDS—PAGE电泳显示约56kDa的目的重组蛋白表达条带，Western—blot分析显示该重组蛋白可与恙虫病病人血清反应，阴性血清则未出现相应印迹，ELISA分析结果显示该蛋白最低浓度为80ng／μL时仍可检出阳性血清；5μg／mL蛋白可检测阳性血清滴度高达1：12800，证实其具有较强反应原性。本实验获得恙虫病东方体合肥株56kDa截短外膜蛋白基因工程抗原具有良好的抗原活性，有望进一步应用于恙虫病的诊断、疫苗研制及致病机制分析。