中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2009年
2期
170-174
,共5页
呼吸道合胞病毒%病毒融合蛋白质类%杆状病毒科%真核细胞%印迹法,蛋白质
呼吸道閤胞病毒%病毒融閤蛋白質類%桿狀病毒科%真覈細胞%印跡法,蛋白質
호흡도합포병독%병독융합단백질류%간상병독과%진핵세포%인적법,단백질
Respiratory syncytial viruses%Viral fusion proteins%Baculoviridae%Eukaryotic cells%Blotting,western
目的 研究人呼吸道合胞病毒(RSV)F基因第546～881碱基编码的融合蛋白(即F蛋白)主要抗原性区域第168～289氨基酸片段的抗原性初步探讨其作为诊断抗原的应用价值.方法 用逆转录(RT)-PCR的方法扩增出RSV Long株F基因546～881 bp片段(F'),将其插入到转移载体质粒pBacPAK9中,获得相应的重组质粒pBacRSV F',与线性杆状病毒BacPAK6 DNA(Bsu36 Ⅰ酶切)共转染Sf9昆虫细胞获得重组病毒BacPAK F',并在昆虫细胞中表达.重组蛋白用Ni2+螫合柱亲和层析纯化,用免疫印迹(Western blot,WB)法检测重组蛋白的抗原活性.并用该方法检测33例急性下呼吸道感染患儿血清特异性抗体,同时用间接免疫荧光法检测患儿的鼻咽分泌物中RSV抗原.对两种方法检测标本的阴性率进行比较.结果 重组病毒BacPAK F'在昆虫细胞中表达相对分子质量约13 000的重组蛋白.纯化后的重组蛋白能与特异性抗RSV抗体结合.WB检测33例急性下呼吸道患儿血清特异性抗体,结果显示阳性11例,阳性率为33.3%,免疫荧光法检测结果阳性9例,阳性率为27.3%,2种检测方法阳性率差异无统计学意义(x2=0.29,P>0.05).结论 纯化后的在昆虫细胞中表达的RSV F基因546～881 bp片段编码的融合蛋白片段具有较强的抗原活性,对RSV感染的快速诊断具有一定的临床应用价值.
目的 研究人呼吸道閤胞病毒(RSV)F基因第546～881堿基編碼的融閤蛋白(即F蛋白)主要抗原性區域第168～289氨基痠片段的抗原性初步探討其作為診斷抗原的應用價值.方法 用逆轉錄(RT)-PCR的方法擴增齣RSV Long株F基因546～881 bp片段(F'),將其插入到轉移載體質粒pBacPAK9中,穫得相應的重組質粒pBacRSV F',與線性桿狀病毒BacPAK6 DNA(Bsu36 Ⅰ酶切)共轉染Sf9昆蟲細胞穫得重組病毒BacPAK F',併在昆蟲細胞中錶達.重組蛋白用Ni2+螫閤柱親和層析純化,用免疫印跡(Western blot,WB)法檢測重組蛋白的抗原活性.併用該方法檢測33例急性下呼吸道感染患兒血清特異性抗體,同時用間接免疫熒光法檢測患兒的鼻嚥分泌物中RSV抗原.對兩種方法檢測標本的陰性率進行比較.結果 重組病毒BacPAK F'在昆蟲細胞中錶達相對分子質量約13 000的重組蛋白.純化後的重組蛋白能與特異性抗RSV抗體結閤.WB檢測33例急性下呼吸道患兒血清特異性抗體,結果顯示暘性11例,暘性率為33.3%,免疫熒光法檢測結果暘性9例,暘性率為27.3%,2種檢測方法暘性率差異無統計學意義(x2=0.29,P>0.05).結論 純化後的在昆蟲細胞中錶達的RSV F基因546～881 bp片段編碼的融閤蛋白片段具有較彊的抗原活性,對RSV感染的快速診斷具有一定的臨床應用價值.
목적 연구인호흡도합포병독(RSV)F기인제546～881감기편마적융합단백(즉F단백)주요항원성구역제168～289안기산편단적항원성초보탐토기작위진단항원적응용개치.방법 용역전록(RT)-PCR적방법확증출RSV Long주F기인546～881 bp편단(F'),장기삽입도전이재체질립pBacPAK9중,획득상응적중조질립pBacRSV F',여선성간상병독BacPAK6 DNA(Bsu36 Ⅰ매절)공전염Sf9곤충세포획득중조병독BacPAK F',병재곤충세포중표체.중조단백용Ni2+석합주친화층석순화,용면역인적(Western blot,WB)법검측중조단백적항원활성.병용해방법검측33례급성하호흡도감염환인혈청특이성항체,동시용간접면역형광법검측환인적비인분비물중RSV항원.대량충방법검측표본적음성솔진행비교.결과 중조병독BacPAK F'재곤충세포중표체상대분자질량약13 000적중조단백.순화후적중조단백능여특이성항RSV항체결합.WB검측33례급성하호흡도환인혈청특이성항체,결과현시양성11례,양성솔위33.3%,면역형광법검측결과양성9례,양성솔위27.3%,2충검측방법양성솔차이무통계학의의(x2=0.29,P>0.05).결론 순화후적재곤충세포중표체적RSV F기인546～881 bp편단편마적융합단백편단구유교강적항원활성,대RSV감염적쾌속진단구유일정적림상응용개치.
Objective To explore the antigenicity of the recombinant respiration syncytial virus (RSV)fusion protein (amino acids 168-289) encoded by 546-881 bases of the fusion gene expressed in insect baculoviruses expression system.Methods The fragment F' of fusion gene 546-881 bases was amplified from viral RNA( Long strain ) by the reverse transcription-polymerase chain reaction(RT-PCR).F' was inserted into transfer vector pBacPAK9 and the recombinant plasmid pBacRSV F' was constructed.Sf9 insect cells were then co-transfeeted with a mixture of recombinant plasmids pBacRSV F' and linearized BacPAK6 viral DNA( Bsu36 Ⅰ -digested).The recombinant baculoviruses BacPAK F' was constructed and was able to express the recombinant protein in Sf9 insect cells.The recombinant protein was purified by Ni2+ NTA chromatography and its antigenicity was identified by Western blot(WB) analysis.Specimens including the nasopharyngeal aspirates(NPAs) and sera were collected from 33 infants and young children with acute lower respiration tract infection. Indirect immunofluorecenee assay (IFA) and WB were used to detect the RSV antigen in NPAs and the anti-RSV antibody in sera respectively.Results The recombinant protein (molecular weight 13 000) was expressed in Sf9 insect cells.WB analysis demonstrated that the purified recombinant protein had a specific RSV antibody-binding activity. The recombinant protein could be recognized by positive serum infected by RSV.The positive rate was 27.3% and 33.3% respectively.There was no significant difference between them(X2 = 0.29 ,P ＞ 0.05).Conclusion The recombinant respiratory syncytial virus fusion gene (546-881 bp) encoding protein is expressed in Sf9 insect cells and it has strong antigenicity and could have clinical application value for detection of RSV infection.
