中国血吸虫病防治杂志
中國血吸蟲病防治雜誌
중국혈흡충병방치잡지
CHINESE JOURNAL OF SCHISTOSOMIASIS CONTROL
2014年
2期
160-164
,共5页
王飞%王晓婷%戴洋%徐颖%邢云天%曲国立%戴建荣
王飛%王曉婷%戴洋%徐穎%邢雲天%麯國立%戴建榮
왕비%왕효정%대양%서영%형운천%곡국립%대건영
日本血吸虫%VCP基因%原核表达%荧光实时定量PCR
日本血吸蟲%VCP基因%原覈錶達%熒光實時定量PCR
일본혈흡충%VCP기인%원핵표체%형광실시정량PCR
Schistosoma japonicum%Valosin-containing protein gene(VCP gene)%Prokaryotical expression%Fluorescence-based quantitative real-time polymerase chain reaction
目的:从原核表达日本血吸虫含缬酪肽蛋白(VCP)基因,并分析其在不同生活史期的mRNA表达水平。方法提取日本血吸虫虫卵RNA,逆转录为cDNA,PCR扩增日本血吸虫VCP基因,亚克隆至原核表达载体pET15b;重组质粒转化入E. coli BL21,异丙基硫代半乳糖苷(IPTG)诱导目的基因表达,并采用包涵体纯化方法获取重组蛋白,产物行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行分析鉴定。提取日本血吸虫尾蚴、童虫、雌虫、雄虫、虫卵RNA,DNase消化,纯化后逆转录为cDNA,采用荧光实时定量PCR分析VCP基因在上述生活史期的表达水平。结果 PCR扩增获得日本血吸虫VCP基因,经重组质粒表达和包涵体纯化成功获得重组蛋白。荧光实时定量PCR检测发现,日本血吸虫VCP基因在尾蚴中的mRNA表达水平最高,在童虫、虫卵、雄虫、雌虫中表达水平较低。结论获得日本血吸虫VCP基因编码的重组蛋白;日本血吸虫VCP基因mRNA在尾蚴阶段表达水平最高,在童虫、虫卵、雄虫、雌虫中表达水平较低。。
目的:從原覈錶達日本血吸蟲含纈酪肽蛋白(VCP)基因,併分析其在不同生活史期的mRNA錶達水平。方法提取日本血吸蟲蟲卵RNA,逆轉錄為cDNA,PCR擴增日本血吸蟲VCP基因,亞剋隆至原覈錶達載體pET15b;重組質粒轉化入E. coli BL21,異丙基硫代半乳糖苷(IPTG)誘導目的基因錶達,併採用包涵體純化方法穫取重組蛋白,產物行十二烷基硫痠鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)進行分析鑒定。提取日本血吸蟲尾蚴、童蟲、雌蟲、雄蟲、蟲卵RNA,DNase消化,純化後逆轉錄為cDNA,採用熒光實時定量PCR分析VCP基因在上述生活史期的錶達水平。結果 PCR擴增穫得日本血吸蟲VCP基因,經重組質粒錶達和包涵體純化成功穫得重組蛋白。熒光實時定量PCR檢測髮現,日本血吸蟲VCP基因在尾蚴中的mRNA錶達水平最高,在童蟲、蟲卵、雄蟲、雌蟲中錶達水平較低。結論穫得日本血吸蟲VCP基因編碼的重組蛋白;日本血吸蟲VCP基因mRNA在尾蚴階段錶達水平最高,在童蟲、蟲卵、雄蟲、雌蟲中錶達水平較低。。
목적:종원핵표체일본혈흡충함힐락태단백(VCP)기인,병분석기재불동생활사기적mRNA표체수평。방법제취일본혈흡충충란RNA,역전록위cDNA,PCR확증일본혈흡충VCP기인,아극륭지원핵표체재체pET15b;중조질립전화입E. coli BL21,이병기류대반유당감(IPTG)유도목적기인표체,병채용포함체순화방법획취중조단백,산물행십이완기류산납-취병희선알응효전영(SDS-PAGE)진행분석감정。제취일본혈흡충미유、동충、자충、웅충、충란RNA,DNase소화,순화후역전록위cDNA,채용형광실시정량PCR분석VCP기인재상술생활사기적표체수평。결과 PCR확증획득일본혈흡충VCP기인,경중조질립표체화포함체순화성공획득중조단백。형광실시정량PCR검측발현,일본혈흡충VCP기인재미유중적mRNA표체수평최고,재동충、충란、웅충、자충중표체수평교저。결론획득일본혈흡충VCP기인편마적중조단백;일본혈흡충VCP기인mRNA재미유계단표체수평최고,재동충、충란、웅충、자충중표체수평교저。。
Objective To prokaryotically express the valosin-containing protein(VCP)of Schistosoma japonicum,and ana-lyze its VCP mRNA expressions in the cercaria,schistosomulum,adult worm(female and male worms)and egg. Methods RNA of S. japonicum eggs were extracted,and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction(PCR),and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells,and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body,and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The RNA(s)of cercaria,schistosomulum,female adult worm,male adult worm,and egg of S. japonicum were extracted,digested with DNase,purified,and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR. Results The VCP gene of S. japonicum was yielded by PCR amplification,and the recombinant pro-tein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR,while low expressions were found in the schistosomulum,egg,female and male adult worms. Conclusion The recombinant protein encoded by the VCP gene of S. ja-ponicum is successfully obtained,and the VCP mRNA expression is determined in various developmental stages of S. japonicum.