CAJ | 학술논문

目的：制备抗人C12orf28多克隆抗体，明确C12orf28编码蛋白在免疫组织中的分布特征。方法选取免疫原性高、特异性强的羧基端257个氨基酸作为目的片段，以人外周血单个核细胞（PBMC）cDNA为模板，采用PCR获得目的基因C12orf28C257；构建其原核表达载体pET32a(+)-C12orf28C257，转化入大肠埃希菌BL21（DE3），异丙基β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白；将重组蛋白免疫大耳白大白兔制备兔源性多克隆抗体，采用ELISA和Western blot分析检测多克隆抗体效价和特异性，采用Western blot明确C12orf28的组织分布特征。结果 PCR扩增得到C12orf28C257目的基因片段，诱导表达重组蛋白并制备了其多克隆抗体。ELISA分析结果显示，多克隆抗体效价＞1︰1.28×106，Western blot分析鉴定得出多克隆抗体具有较高的特异性。结论未知功能基因C12orf28在胎儿肠、淋巴、胸腺和心肌等组织中均有不同程度的表达。
목적：제비항인C12orf28다극륭항체，명학C12orf28편마단백재면역조직중적분포특정。방법선취면역원성고、특이성강적최기단257개안기산작위목적편단，이인외주혈단개핵세포（PBMC）cDNA위모판，채용PCR획득목적기인C12orf28C257；구건기원핵표체재체pET32a(+)-C12orf28C257，전화입대장애희균BL21（DE3），이병기β-D-류대필남반유당감（IPTG）유도표체중조단백；장중조단백면역대이백대백토제비토원성다극륭항체，채용ELISA화Western blot분석검측다극륭항체효개화특이성，채용Western blot명학C12orf28적조직분포특정。결과 PCR확증득도C12orf28C257목적기인편단，유도표체중조단백병제비료기다극륭항체。ELISA분석결과현시，다극륭항체효개＞1︰1.28×106，Western blot분석감정득출다극륭항체구유교고적특이성。결론미지공능기인C12orf28재태인장、림파、흉선화심기등조직중균유불동정도적표체。
Objective To prepare the polyclonal antibody of C12orf28 and observe the expression features of C12orf28 in immunologic tissues. Methods The peptide sequence with high immunogenicity and specificity that contains 257 amino acids of the carboxy-terminal was selected and amplified by reverse transcription PCR. The DNA fragment C12orf28C257 was inserted into pET32a(+) plasmid and transformed into Escherichia coli BL21. The protein expression was induced with IPTG in vitro. Anti-C12orf28 was prepared by immunizing the big ears rabbit and was analyzed by Western blot and ELISA. The distribution of C12orf28 in tissues was detected by Western blot. Results The DNA fragment C12orf28C257 was cloned and its recombinant protein was successfully induced. The antibody titer was more than 1︰1.28 × 106 by ELISA and the high specificity of the polyclonal antibody was detected by Western blot. Conclusion The new gene C12orf28 is expressed in the fetal tissues such as intestine, lymph node, thymus and myocardiun, etc.