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
2012年
6期
456-459
,共4页
赵艳丰%闫梁%许斌%贲海静%杨金华%周双成%瞿涤
趙豔豐%閆樑%許斌%賁海靜%楊金華%週雙成%瞿滌
조염봉%염량%허빈%분해정%양금화%주쌍성%구조
膜联蛋白2%多克隆抗体%肝炎病毒,乙型,鸭
膜聯蛋白2%多剋隆抗體%肝炎病毒,乙型,鴨
막련단백2%다극륭항체%간염병독,을형,압
Annexin A2%Polyclonal antibody%Hepatitis B virus,duck
目的 克隆樱桃谷鸭膜联蛋白2(Annexin A2)并制备抗体,用于鸭Annexin A2蛋白的表达分析.方法 采用RT-PCR方法从鸭肝脏组织中克隆鸭Annexin A2 cDNA序列,将该cDNA序列亚克隆到原核表达载体pET-28a(+),构建重组原核表达载体pET-28a(+)-Annexin A2.异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导Annexin A2重组质粒原核表达,应用镍柱纯化Annexin A2融合蛋白.腹腔注射Annexin A2融合蛋白免疫小鼠,采用免疫印迹鉴定小鼠抗鸭Annexin A2多克隆抗体并检测鸭原代肝细胞在培养12、24、72 h以及120 h Annexin A2蛋白表达情况.结果 成功构建了原核表达载体pET-28a(+)-Annexin A2,经NcoI和XhoI双酶切,琼脂糖凝胶电泳显示5369 bp和1020 bp条带,重组质粒测序结果显示重组序列正确.IPTG诱导重组质粒表达产生相对分子质量为36 000的可溶性融合蛋白.Annexin A2融合蛋白免疫小鼠,获得的抗血清1∶2000稀释经免疫印迹仍能检测到靶蛋白,获得的小鼠抗鸭Annexin A2多克隆抗体可应用于鸭原代肝细胞表达检测,Annexin A2蛋白在鸭原代肝细胞培养12、24、72 h以及120 h均有表达.结论 制备了小鼠抗鸭Annexin A2多克隆抗体,可为探讨Annexin A2影响鸭乙型肝炎病毒感染分子机制提供实验依据.
目的 剋隆櫻桃穀鴨膜聯蛋白2(Annexin A2)併製備抗體,用于鴨Annexin A2蛋白的錶達分析.方法 採用RT-PCR方法從鴨肝髒組織中剋隆鴨Annexin A2 cDNA序列,將該cDNA序列亞剋隆到原覈錶達載體pET-28a(+),構建重組原覈錶達載體pET-28a(+)-Annexin A2.異丙基-β-D-硫代吡喃半乳糖苷(IPTG)誘導Annexin A2重組質粒原覈錶達,應用鎳柱純化Annexin A2融閤蛋白.腹腔註射Annexin A2融閤蛋白免疫小鼠,採用免疫印跡鑒定小鼠抗鴨Annexin A2多剋隆抗體併檢測鴨原代肝細胞在培養12、24、72 h以及120 h Annexin A2蛋白錶達情況.結果 成功構建瞭原覈錶達載體pET-28a(+)-Annexin A2,經NcoI和XhoI雙酶切,瓊脂糖凝膠電泳顯示5369 bp和1020 bp條帶,重組質粒測序結果顯示重組序列正確.IPTG誘導重組質粒錶達產生相對分子質量為36 000的可溶性融閤蛋白.Annexin A2融閤蛋白免疫小鼠,穫得的抗血清1∶2000稀釋經免疫印跡仍能檢測到靶蛋白,穫得的小鼠抗鴨Annexin A2多剋隆抗體可應用于鴨原代肝細胞錶達檢測,Annexin A2蛋白在鴨原代肝細胞培養12、24、72 h以及120 h均有錶達.結論 製備瞭小鼠抗鴨Annexin A2多剋隆抗體,可為探討Annexin A2影響鴨乙型肝炎病毒感染分子機製提供實驗依據.
목적 극륭앵도곡압막련단백2(Annexin A2)병제비항체,용우압Annexin A2단백적표체분석.방법 채용RT-PCR방법종압간장조직중극륭압Annexin A2 cDNA서렬,장해cDNA서렬아극륭도원핵표체재체pET-28a(+),구건중조원핵표체재체pET-28a(+)-Annexin A2.이병기-β-D-류대필남반유당감(IPTG)유도Annexin A2중조질립원핵표체,응용얼주순화Annexin A2융합단백.복강주사Annexin A2융합단백면역소서,채용면역인적감정소서항압Annexin A2다극륭항체병검측압원대간세포재배양12、24、72 h이급120 h Annexin A2단백표체정황.결과 성공구건료원핵표체재체pET-28a(+)-Annexin A2,경NcoI화XhoI쌍매절,경지당응효전영현시5369 bp화1020 bp조대,중조질립측서결과현시중조서렬정학.IPTG유도중조질립표체산생상대분자질량위36 000적가용성융합단백.Annexin A2융합단백면역소서,획득적항혈청1∶2000희석경면역인적잉능검측도파단백,획득적소서항압Annexin A2다극륭항체가응용우압원대간세포표체검측,Annexin A2단백재압원대간세포배양12、24、72 h이급120 h균유표체.결론 제비료소서항압Annexin A2다극륭항체,가위탐토Annexin A2영향압을형간염병독감염분자궤제제공실험의거.
Objective To clone Annexin A2,the protein isolated from Cherry Valley duck,and to prepare the antibody for subsequent expression analysis.Methods The cDNA sequence of duck Annexin A2 from duck liver tissue was amplified by reverse transeription polymerase chain reaction and was subcloned into the prokaryotic expression vector pET-28a (+) for construction of pET-28a (+)-Annexin A2,a recombinant plasmid,followed by in situ expression induced by isopropryl-β-D-thiogalactoside (IPTG)and purification of Annexin A2 protein by using nickel columns.The mice were challenged with Annexin A2 via peritoneal injection.The Annexin A2 polyclonal antibodies were identified by Western blotting and the expression in primary duck hepatic cells was assayed at hours 12,24,72 and 120 respectively.Results Upon successful construction of prokaryocyte recombinant plasmid pET-28a(+)-Annexin A2,the sequence was proven by Ncol and Xhol doubledigestion followed by agar electrophoresis showing 2 characteristic bands,namely,5369 bp and 1020 bp,and Recombinant plasmid sequencing results showed that recombinant correct sequence.A soluble 36 000 fusion protein was detected by IPTC induction.Following Annexin A2 fusion protein challenge,the target protein in the 1 ∶ 2000 diluted anti-serum could be assayed via Western blotting.The polyclonal antibodies obtained from mice could be adopted for detection of duck hepatic cell surface Annexin A2,which was expressed in cultured primary hepatic cells at hours 12,24,72 and 120 respectively.Conclusion The preparation of mice polyclonal against duck Annexin A2 may offer rationale for the molecular mechanisms in duck hepatitis B infection.
