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
2009年
10期
890-893
,共4页
黄吉城%武婕%郭波旋%张宏斌%相大鹏%师永霞%李小波%洪烨%郑夔%幸芦琴%杨晓
黃吉城%武婕%郭波鏇%張宏斌%相大鵬%師永霞%李小波%洪燁%鄭夔%倖蘆琴%楊曉
황길성%무첩%곽파선%장굉빈%상대붕%사영하%리소파%홍엽%정기%행호금%양효
人禽流感病毒H5N1%人源化单链抗体%噬菌体文库%构建%筛选
人禽流感病毒H5N1%人源化單鏈抗體%噬菌體文庫%構建%篩選
인금류감병독H5N1%인원화단련항체%서균체문고%구건%사선
Human H5N1 virus%Humanized single chain antibody%Phage library%Construction%Screening
目的 利用抗人禽流感病毒H5N1 IgG抗体阳性的人禽流感康复患者外周血淋巴细胞,构建人源化Fv段单链抗体(seFv)噬菌体文库,并筛选与禽流感病毒相关蛋白有结合活性的scFv抗体文库.方法 提取人外周血淋巴细胞总RNA,逆转录成cDNA,以其为模板,利用家族特异性IgG基因的引物,扩增重链和轻链的可变区基因,并用合成的连接子将轻链和重链基因连接成单链抗体片段后,重组到噬菌粒载体pCANTAB5E中.将重组噬菌粒载体电转化大肠杆菌TG1,酶切和PCR鉴定抗体库的重组率,通过测定噬菌体抗体库的滴度计算抗体库的库容,用特异性禽流感病毒相关蛋白筛选表达的单链抗体.结果 构建了源于人禽流感康复患者血清的scFv抗体文库,库容为3.75×104;筛选出与禽流感病毒相关蛋白有结合活性的scFv抗体文库.结论 成功构建了抗人禽流感病毒H5N1的人源scFv噬菌体抗体库,并筛选出特异性结合人禽流感病毒相关蛋白的单链抗体,为进一步制备快速检测试剂和治疗研究提供了基础数据.
目的 利用抗人禽流感病毒H5N1 IgG抗體暘性的人禽流感康複患者外週血淋巴細胞,構建人源化Fv段單鏈抗體(seFv)噬菌體文庫,併篩選與禽流感病毒相關蛋白有結閤活性的scFv抗體文庫.方法 提取人外週血淋巴細胞總RNA,逆轉錄成cDNA,以其為模闆,利用傢族特異性IgG基因的引物,擴增重鏈和輕鏈的可變區基因,併用閤成的連接子將輕鏈和重鏈基因連接成單鏈抗體片段後,重組到噬菌粒載體pCANTAB5E中.將重組噬菌粒載體電轉化大腸桿菌TG1,酶切和PCR鑒定抗體庫的重組率,通過測定噬菌體抗體庫的滴度計算抗體庫的庫容,用特異性禽流感病毒相關蛋白篩選錶達的單鏈抗體.結果 構建瞭源于人禽流感康複患者血清的scFv抗體文庫,庫容為3.75×104;篩選齣與禽流感病毒相關蛋白有結閤活性的scFv抗體文庫.結論 成功構建瞭抗人禽流感病毒H5N1的人源scFv噬菌體抗體庫,併篩選齣特異性結閤人禽流感病毒相關蛋白的單鏈抗體,為進一步製備快速檢測試劑和治療研究提供瞭基礎數據.
목적 이용항인금류감병독H5N1 IgG항체양성적인금류감강복환자외주혈림파세포,구건인원화Fv단단련항체(seFv)서균체문고,병사선여금류감병독상관단백유결합활성적scFv항체문고.방법 제취인외주혈림파세포총RNA,역전록성cDNA,이기위모판,이용가족특이성IgG기인적인물,확증중련화경련적가변구기인,병용합성적련접자장경련화중련기인련접성단련항체편단후,중조도서균립재체pCANTAB5E중.장중조서균립재체전전화대장간균TG1,매절화PCR감정항체고적중조솔,통과측정서균체항체고적적도계산항체고적고용,용특이성금류감병독상관단백사선표체적단련항체.결과 구건료원우인금류감강복환자혈청적scFv항체문고,고용위3.75×104;사선출여금류감병독상관단백유결합활성적scFv항체문고.결론 성공구건료항인금류감병독H5N1적인원scFv서균체항체고,병사선출특이성결합인금류감병독상관단백적단련항체,위진일보제비쾌속검측시제화치료연구제공료기출수거.
Objective To construct and screen the phage antibody library for seFv fragment from recovery phase patient infected with H5N1 virus.Methods Total RNA was extractod from peripheral blood lymphocytes from a patient infected with H5N1 virus and was transformed to cDNA by reverse transcription.The variable domains of heavy and light chains were amplified by PCR with a set of specific primer of human IgG.The scFv gene fragments linked VH and VL with synthesized scFv linker were cloned into phagemid pCANTAB5E vector.Then the recombined phagemids were electroperated into E.coli TG1.The recombinant rate of scFv phage antibody library Was identified by enzyme digestion analysis and PCR.And the library repertoire Was determined by titration the PFU of library.Results The phage antibody library of scFv which specific binding to the proteins of H5N1 virus Was generated from the peripheral blood of the recovery phase patient infected with H5N1 virus and the repertoire of library was 3.75×104.Conclusion Phage antibody library of humanized scFv fragment which specific binding to the proteins of H5N1 virus has been constructed and screened successfully,which lay the foundation for further study of quick detection method and medicihal purpose.
