中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2008年
9期
782-786
,共5页
田新贵%周荣%李海涛%龚四堂%张其威%朱冰%盛慧英%钟家禹
田新貴%週榮%李海濤%龔四堂%張其威%硃冰%盛慧英%鐘傢禹
전신귀%주영%리해도%공사당%장기위%주빙%성혜영%종가우
诺如病毒衣壳蛋白%人3型腺病毒%同源重组
諾如病毒衣殼蛋白%人3型腺病毒%同源重組
낙여병독의각단백%인3형선병독%동원중조
Norovirus capsid protein%Human adenovirus type 3%Homologous recombination
目的 制备表达诺如病毒衣壳蛋白的重组人3型腺病毒.方法 将诺如病毒衣壳蛋白基因(Noro-orf2)克隆到腺病毒穿梭载体pBSE3CMV-egfp上,与线性化人3型腺病毒骨架质粒pBRAdv3共电转化感受态大肠杆菌BJ5183,使其在细菌内发生同源重组,带Noro-orf2基因的表达框置换腺病毒E3区,PCR及酶切筛选得到重组腺病毒质粒,将重组腺病毒质粒转染Hep-2细胞进行包装,获得感染性的重组腺病毒粒子,免疫组化分析重组腺病毒中诺如病毒衣壳蛋白的表达.结果 同源重组后经酶切和PCR鉴定证明插入Noro-orf2基因的重组腺病毒质粒pBRAdv3E3dNor成功构建,并经转染包装得到高滴度的重组腺病毒Adv3E3dNor,免疫组化证明诺如病毒衣壳蛋白得到表达.结论 成功构建表达诺如病毒衣壳蛋白的重组3型腺病毒Adv3E3dNor,为研制人3型腺病毒-诺如病毒双价疫苗奠定了基础.
目的 製備錶達諾如病毒衣殼蛋白的重組人3型腺病毒.方法 將諾如病毒衣殼蛋白基因(Noro-orf2)剋隆到腺病毒穿梭載體pBSE3CMV-egfp上,與線性化人3型腺病毒骨架質粒pBRAdv3共電轉化感受態大腸桿菌BJ5183,使其在細菌內髮生同源重組,帶Noro-orf2基因的錶達框置換腺病毒E3區,PCR及酶切篩選得到重組腺病毒質粒,將重組腺病毒質粒轉染Hep-2細胞進行包裝,穫得感染性的重組腺病毒粒子,免疫組化分析重組腺病毒中諾如病毒衣殼蛋白的錶達.結果 同源重組後經酶切和PCR鑒定證明插入Noro-orf2基因的重組腺病毒質粒pBRAdv3E3dNor成功構建,併經轉染包裝得到高滴度的重組腺病毒Adv3E3dNor,免疫組化證明諾如病毒衣殼蛋白得到錶達.結論 成功構建錶達諾如病毒衣殼蛋白的重組3型腺病毒Adv3E3dNor,為研製人3型腺病毒-諾如病毒雙價疫苗奠定瞭基礎.
목적 제비표체낙여병독의각단백적중조인3형선병독.방법 장낙여병독의각단백기인(Noro-orf2)극륭도선병독천사재체pBSE3CMV-egfp상,여선성화인3형선병독골가질립pBRAdv3공전전화감수태대장간균BJ5183,사기재세균내발생동원중조,대Noro-orf2기인적표체광치환선병독E3구,PCR급매절사선득도중조선병독질립,장중조선병독질립전염Hep-2세포진행포장,획득감염성적중조선병독입자,면역조화분석중조선병독중낙여병독의각단백적표체.결과 동원중조후경매절화PCR감정증명삽입Noro-orf2기인적중조선병독질립pBRAdv3E3dNor성공구건,병경전염포장득도고적도적중조선병독Adv3E3dNor,면역조화증명낙여병독의각단백득도표체.결론 성공구건표체낙여병독의각단백적중조3형선병독Adv3E3dNor,위연제인3형선병독-낙여병독쌍개역묘전정료기출.
Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.
