中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2013年
3期
204-209
,共6页
范莉%姜红叶%李锦波%陈淑琴
範莉%薑紅葉%李錦波%陳淑琴
범리%강홍협%리금파%진숙금
防御素2%人%重组质粒
防禦素2%人%重組質粒
방어소2%인%중조질립
Defensin-2%Human%Recombinant plasmid
目的 构建人β防御素2(hBD-2)的真核表达载体PLXSN的重组质粒pLXSN-hBD2,将其转染至大鼠子宫内膜异位症(EMS)模型的局部异位病灶组织,并检测hBD-2基因和蛋白表达.方法 利用双酶切定位定向克隆技术,将hBD-2基因片段连接于pLXSN质粒的EcoR Ⅰ和BamH Ⅰ两个酶切位点之间,构建重组质粒pLXSN-hBD2.自体内膜移植法建立大鼠EMS模型,将pLXSN-hBD2局部多点注射至其局部异位病灶组织,Western印迹检测局部病灶组织中的hBD-2蛋白表达.结果 经过重组质粒的鉴定电泳以及测序鉴定,设计合成的基因片段序列与GenBank中hBD-2cDNA序列完全相同,其中碱基54-248为目的基因hBD-2序列.Western印迹证实大鼠EMS模型异位病灶局部有hBD-2蛋白的表达.结论 通过局部多点注射的方法,可将携带hBD-2基因的重组质粒pLXSN-hBD2成功转染至大鼠EMS异位病灶组织中,并获得hBD-2蛋白的表达.
目的 構建人β防禦素2(hBD-2)的真覈錶達載體PLXSN的重組質粒pLXSN-hBD2,將其轉染至大鼠子宮內膜異位癥(EMS)模型的跼部異位病竈組織,併檢測hBD-2基因和蛋白錶達.方法 利用雙酶切定位定嚮剋隆技術,將hBD-2基因片段連接于pLXSN質粒的EcoR Ⅰ和BamH Ⅰ兩箇酶切位點之間,構建重組質粒pLXSN-hBD2.自體內膜移植法建立大鼠EMS模型,將pLXSN-hBD2跼部多點註射至其跼部異位病竈組織,Western印跡檢測跼部病竈組織中的hBD-2蛋白錶達.結果 經過重組質粒的鑒定電泳以及測序鑒定,設計閤成的基因片段序列與GenBank中hBD-2cDNA序列完全相同,其中堿基54-248為目的基因hBD-2序列.Western印跡證實大鼠EMS模型異位病竈跼部有hBD-2蛋白的錶達.結論 通過跼部多點註射的方法,可將攜帶hBD-2基因的重組質粒pLXSN-hBD2成功轉染至大鼠EMS異位病竈組織中,併穫得hBD-2蛋白的錶達.
목적 구건인β방어소2(hBD-2)적진핵표체재체PLXSN적중조질립pLXSN-hBD2,장기전염지대서자궁내막이위증(EMS)모형적국부이위병조조직,병검측hBD-2기인화단백표체.방법 이용쌍매절정위정향극륭기술,장hBD-2기인편단련접우pLXSN질립적EcoR Ⅰ화BamH Ⅰ량개매절위점지간,구건중조질립pLXSN-hBD2.자체내막이식법건립대서EMS모형,장pLXSN-hBD2국부다점주사지기국부이위병조조직,Western인적검측국부병조조직중적hBD-2단백표체.결과 경과중조질립적감정전영이급측서감정,설계합성적기인편단서렬여GenBank중hBD-2cDNA서렬완전상동,기중감기54-248위목적기인hBD-2서렬.Western인적증실대서EMS모형이위병조국부유hBD-2단백적표체.결론 통과국부다점주사적방법,가장휴대hBD-2기인적중조질립pLXSN-hBD2성공전염지대서EMS이위병조조직중,병획득hBD-2단백적표체.
Objective To construct pLXSN-hBD2,the human defensin-2 (hBD-2) eukaryotic pLXSN recombinant plasmid,thus allowing for transfection to the ectopic loci of endometriosis in rats (EMS) and subsequent assessment of hBD-2 gene and protein expression.Methods The hBD-2 gene fragment was linked to the digestion sites of EcoR Ⅰ and BamH Ⅰ of the pLXSN plasmid for construction of the recombinant plasmid pLXSN-hBD2 by using double enzyme positioning and directional cloning technology.The EMS rat model was constructed by autologous membrane transplantation.This was followed by multi-foci injection of pLXSN-hBD2 to the ectopic foci,thus allowing for determination of hBD2 protein expression in ectopic endometrium by Western blotting.Results Following recombinant plasmid electrophoresis and sequencing,the designed gene fragment possessed an identical sequence with that of hBD-2 cDNA in GenBank,in which the 54-248 base pairs were consistent with the sequence of hBD-2 target gene.The expression of hBD-2 protein was observed in ectopic foci,as evidenced by Western blotting.Conclusion By multi-foci injection,the recombinant plasmid pLXSN-hBD2 containing hBD-2 gene can be successfully transfected to the endometriosis tissues in rats for harvesting hBD-2 protein.
