中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2010年
1期
27-29
,共3页
王乃福%马晶%张晓光%张晓梅%白天%王敏%温乐英%王大燕%舒跃龙%周玲%曾毅
王迺福%馬晶%張曉光%張曉梅%白天%王敏%溫樂英%王大燕%舒躍龍%週玲%曾毅
왕내복%마정%장효광%장효매%백천%왕민%온악영%왕대연%서약룡%주령%증의
流感病毒A型%核蛋白类%蛋白质相互作用
流感病毒A型%覈蛋白類%蛋白質相互作用
류감병독A형%핵단백류%단백질상호작용
Influenza A virus%Nucleoproteins%Proteininteraction mapping
目的 原核表达并纯化禽流感病毒A/Anhui/1/2005(H5N1)的NP蛋白,并筛选人支气管上皮BEAS-2B细胞总蛋白中能够与纯化的NP蛋白相互作用的蛋白.方法 一方面,构建了含有NP基因的原核表达质粒pET30a-NP,并在大肠埃希菌中获得了可溶性表达.亲和层析和离子交换层析两步对NP蛋白进行纯化.另一方面,制备了BEAS-2B细胞总蛋白.在此基础上,联合应用Pull-down与LC-MS/MS技术来筛选并确认细胞中与纯化的NP蛋白相互作用的成分.结果 构建的pET30a-NP质粒在IPTG诱导下在原核细胞中实现了可溶表达,经过两步纯化后,得到可溶的NP蛋白纯品.Pull-down与LC-MS/MS技术初步筛选到BEAS-2B细胞中20个可能与NP蛋白相互作用的细胞蛋白.还需要进一步的实验来验证他们和NP蛋白之间的相互作用.结论 获得了高纯度的可溶NP蛋白及筛选到20个可能与其相互作用的BEAS-2B细胞候选蛋白.
目的 原覈錶達併純化禽流感病毒A/Anhui/1/2005(H5N1)的NP蛋白,併篩選人支氣管上皮BEAS-2B細胞總蛋白中能夠與純化的NP蛋白相互作用的蛋白.方法 一方麵,構建瞭含有NP基因的原覈錶達質粒pET30a-NP,併在大腸埃希菌中穫得瞭可溶性錶達.親和層析和離子交換層析兩步對NP蛋白進行純化.另一方麵,製備瞭BEAS-2B細胞總蛋白.在此基礎上,聯閤應用Pull-down與LC-MS/MS技術來篩選併確認細胞中與純化的NP蛋白相互作用的成分.結果 構建的pET30a-NP質粒在IPTG誘導下在原覈細胞中實現瞭可溶錶達,經過兩步純化後,得到可溶的NP蛋白純品.Pull-down與LC-MS/MS技術初步篩選到BEAS-2B細胞中20箇可能與NP蛋白相互作用的細胞蛋白.還需要進一步的實驗來驗證他們和NP蛋白之間的相互作用.結論 穫得瞭高純度的可溶NP蛋白及篩選到20箇可能與其相互作用的BEAS-2B細胞候選蛋白.
목적 원핵표체병순화금류감병독A/Anhui/1/2005(H5N1)적NP단백,병사선인지기관상피BEAS-2B세포총단백중능구여순화적NP단백상호작용적단백.방법 일방면,구건료함유NP기인적원핵표체질립pET30a-NP,병재대장애희균중획득료가용성표체.친화층석화리자교환층석량보대NP단백진행순화.령일방면,제비료BEAS-2B세포총단백.재차기출상,연합응용Pull-down여LC-MS/MS기술래사선병학인세포중여순화적NP단백상호작용적성분.결과 구건적pET30a-NP질립재IPTG유도하재원핵세포중실현료가용표체,경과량보순화후,득도가용적NP단백순품.Pull-down여LC-MS/MS기술초보사선도BEAS-2B세포중20개가능여NP단백상호작용적세포단백.환수요진일보적실험래험증타문화NP단백지간적상호작용.결론 획득료고순도적가용NP단백급사선도20개가능여기상호작용적BEAS-2B세포후선단백.
Objective To express and purify H5N1 influenza virus (A/Anhui/1/2005) NP in prokaryotic system and to explore the NP-interacting proteins of human bronchial epithelial cells BEAS-2B in vitro . Methods The full length H5N1 NP gene fragment was amplified by PCR, inserted into prokaryotic expression vector(pET30a) to generate NP expression plasmid pET30a-NP. After transforming pET30a-NP into E. coli (BL21), the expression of soluble NP protein was induced by IPTG. The expressed NP protein was purified by two steps with metal chelation chromatography and ion exchange chromatography. Then the total proteins of BEAS-2B cells was extracted for screening the components which have protein-protein interaction with purified NP by pull-down and LC-MS/MS methods. Results The expression of H5N1 NP protein could be induced by IPTG in bacterial system using expression plasmid pET30a-NP. The soluble NP was purified. Twenty proteins were found by pull-down and LC-MS/MS, the further experiments may be needed to prove protein-protein interaction between them. Conclusion The soluble H5N1 NP fusion protein with high purity was obtained and twenty proteins were found which could interact with it by pull-down and LC-MS/MS.
