中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2009年
12期
826-830
,共5页
元艳宏%康佳蕊%闫荣%程虹%庄逢源%王月丹%戴克胜
元豔宏%康佳蕊%閆榮%程虹%莊逢源%王月丹%戴剋勝
원염굉%강가예%염영%정홍%장봉원%왕월단%대극성
血小板%血小板糖蛋白GPIb-IX复合物%磷酸丝氨酸%抗体
血小闆%血小闆糖蛋白GPIb-IX複閤物%燐痠絲氨痠%抗體
혈소판%혈소판당단백GPIb-IX복합물%린산사안산%항체
Blood platelets%Platelet glycoprotein GPIb-IX complex%Phosphoserine%Antibodies
目的 制备兔抗人血小板膜糖蛋白(GP)Ibα C端557～561氨基酸序列多肽的多克隆抗体,并初步用于人血小板GPIbα C端559位丝氨酸磷酸化状态的检测.方法 应用化学方法合成C-R-G-S-L-P(559位丝氨酸非磷酸化,Ser559)和C-R-G-s(p)-L-P(559位丝氨酸磷酸化,pSer559)多肽.将2种多肽分别与钥孔蜮血蓝蛋白交联后,以皮下注射法分别免疫新西兰大白兔,分离获得2种抗血清(抗Ser559多抗和抗pSer559多抗).应用斑点印迹和酶联免疫吸附试验(ELISA)方法 对抗血清进行鉴定并检测效价.从人血小板裂解液中分离纯化血小板GPIbα,利用抗Ser559多抗和抗pSer559多抗、采用ELISA方法检测人血小板GPIbαC端559位丝氨酸磷酸化状况.结果 所制备的2种多抗分别特异性识别各自抗原,效价分别为1:32 000和1:64 000.2种多抗均可与纯化的人血小板GPIbα特异结合,表明人血小板GPIbα 559位点丝氨酸存在磷酸化与非磷酸化两种状态.结论 应用人工合成多肽成功制备出2种可特异性识别丝氨酸磷酸化状态的兔抗人血小板GPIbα胞内段多肽多抗,并证明人血小板GPIbαC端559位丝氨酸存在磷酸化状态.
目的 製備兔抗人血小闆膜糖蛋白(GP)Ibα C耑557～561氨基痠序列多肽的多剋隆抗體,併初步用于人血小闆GPIbα C耑559位絲氨痠燐痠化狀態的檢測.方法 應用化學方法閤成C-R-G-S-L-P(559位絲氨痠非燐痠化,Ser559)和C-R-G-s(p)-L-P(559位絲氨痠燐痠化,pSer559)多肽.將2種多肽分彆與鑰孔蜮血藍蛋白交聯後,以皮下註射法分彆免疫新西蘭大白兔,分離穫得2種抗血清(抗Ser559多抗和抗pSer559多抗).應用斑點印跡和酶聯免疫吸附試驗(ELISA)方法 對抗血清進行鑒定併檢測效價.從人血小闆裂解液中分離純化血小闆GPIbα,利用抗Ser559多抗和抗pSer559多抗、採用ELISA方法檢測人血小闆GPIbαC耑559位絲氨痠燐痠化狀況.結果 所製備的2種多抗分彆特異性識彆各自抗原,效價分彆為1:32 000和1:64 000.2種多抗均可與純化的人血小闆GPIbα特異結閤,錶明人血小闆GPIbα 559位點絲氨痠存在燐痠化與非燐痠化兩種狀態.結論 應用人工閤成多肽成功製備齣2種可特異性識彆絲氨痠燐痠化狀態的兔抗人血小闆GPIbα胞內段多肽多抗,併證明人血小闆GPIbαC耑559位絲氨痠存在燐痠化狀態.
목적 제비토항인혈소판막당단백(GP)Ibα C단557～561안기산서렬다태적다극륭항체,병초보용우인혈소판GPIbα C단559위사안산린산화상태적검측.방법 응용화학방법합성C-R-G-S-L-P(559위사안산비린산화,Ser559)화C-R-G-s(p)-L-P(559위사안산린산화,pSer559)다태.장2충다태분별여약공역혈람단백교련후,이피하주사법분별면역신서란대백토,분리획득2충항혈청(항Ser559다항화항pSer559다항).응용반점인적화매련면역흡부시험(ELISA)방법 대항혈청진행감정병검측효개.종인혈소판렬해액중분리순화혈소판GPIbα,이용항Ser559다항화항pSer559다항、채용ELISA방법검측인혈소판GPIbαC단559위사안산린산화상황.결과 소제비적2충다항분별특이성식별각자항원,효개분별위1:32 000화1:64 000.2충다항균가여순화적인혈소판GPIbα특이결합,표명인혈소판GPIbα 559위점사안산존재린산화여비린산화량충상태.결론 응용인공합성다태성공제비출2충가특이성식별사안산린산화상태적토항인혈소판GPIbα포내단다태다항,병증명인혈소판GPIbαC단559위사안산존재린산화상태.
Objective To prepare rabbit polyclonal antibodies against intraceUular peptides of human platelet glycoprotein GPIbα. Methods Two peptides corresponding to human platelet GPIbα C-terminus were synthesized and purified by high-performance liquid chromatography (HPLC). The peptides were cross-linked with keyhole limpet hemocyanin (KLH). Two New Zealand white rabbits were immunized with conjugated peptides for 3 times. The polyclonal antibodies were purified by Ammonium Sulfate Precipitation and identified by dot blotting and ELISA. GPIbot intracellular poptides phospharylation was tested with these polyclonal antibodies by ELISA. Results The titers of the two polyclonal antibodies against the GPIbα C-terminus poptides were 1 : 32 000 and 1 : 64 000 respectively and both of these antibodies reacted with purified GPlbot. Conclusions Two rabbit polyclonal antibodies against C-terminal peptides of human platelet GPIbα have been prepared successfully, providing a way for the preparation of these kinds of antibody. Both phosphorylation and dephosphorylation states exist in the intracellular poptide of human platelets.
