CAJ | 학술논문

目的：采用多肽制备技术和免疫学技术制备抗钠泵α2亚单位截断性片段及其抗体，为检测和研究钠泵α2亚单位的组织学分布及其功能研究提供实验基础。方法根据NCBI-Genebank获得大鼠钠泵α2亚单位M1~M2膜外区截断性片段目的多肽（LAAMEDEPSNDN），采用9-氟甲氧羰基（Fmoc）固相合成法合成目的多肽，并采用碳化二亚胺法制备出多肽与孔戚血蓝素复合物，免疫新西兰大白兔，免疫4次后获得抗血清，测定其效价。随后采用protein A纯化IgG抗体，并将其用于大鼠主动脉血管平滑肌细胞钠泵α2亚单的组织学检测。结果（1）人工合成的大鼠钠泵α2亚单位截断性片段长13氨基酸残基（LAAMEDEPSNDN-C），理论相对分子质量：1408.48，质谱实测相对分子质量：1407.90；高效色谱分析纯度HPLC纯度>85.5%；（2）ELISA法检测免疫后兔子的抗血清效价均大于1∶512000，蛋白A亲和纯化获得亲和纯化抗体浓度为0.965 mg/ml，按照1∶1000稀释（终浓度1μg/ml），ELISA检测抗体效价为1∶256000；（3）该抗体按照1∶100~1∶200稀释可用于免疫细胞学检测。结论成功制备高效价抗钠泵α2亚单位截断性片段抗体可用于钠泵α2亚单位的ELISA和免疫细胞化学实验，为钠泵α2亚单位的组织细胞学检测和功能研究提供新的实验基础。
목적：채용다태제비기술화면역학기술제비항납빙α2아단위절단성편단급기항체，위검측화연구납빙α2아단위적조직학분포급기공능연구제공실험기출。방법근거NCBI-Genebank획득대서납빙α2아단위M1~M2막외구절단성편단목적다태（LAAMEDEPSNDN），채용9-불갑양탄기（Fmoc）고상합성법합성목적다태，병채용탄화이아알법제비출다태여공척혈람소복합물，면역신서란대백토，면역4차후획득항혈청，측정기효개。수후채용protein A순화IgG항체，병장기용우대서주동맥혈관평활기세포납빙α2아단적조직학검측。결과（1）인공합성적대서납빙α2아단위절단성편단장13안기산잔기（LAAMEDEPSNDN-C），이론상대분자질량：1408.48，질보실측상대분자질량：1407.90；고효색보분석순도HPLC순도>85.5%；（2）ELISA법검측면역후토자적항혈청효개균대우1∶512000，단백A친화순화획득친화순화항체농도위0.965 mg/ml，안조1∶1000희석（종농도1μg/ml），ELISA검측항체효개위1∶256000；（3）해항체안조1∶100~1∶200희석가용우면역세포학검측。결론성공제비고효개항납빙α2아단위절단성편단항체가용우납빙α2아단위적ELISA화면역세포화학실험，위납빙α2아단위적조직세포학검측화공능연구제공신적실험기출。
Objective To prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment (NKAα2 EM1) for studying the pathogenesis of hypertension. Methods According to the GenBank data, the amino acid sequence of NKAα2 EM1 was obtained and the target peptide (LAAMEDEPSNDN) was synthesized using a peptide synthesizer with Fmoc method and purified with high-performance liquid chromatography. The synthesized peptide was then coupled to KLH for immunizing New Zealand white rabbits for 4 times to obtain the antiserum. The IgG antibodies against the synthetic peptide, after affinity purification with Protein A, were used for detecting NKAα2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme- linked immunosorbent assay and immunocytochemistry (ICC). Results The synthesized peptide fragment , which consisted of 13 amino acid residues including one derivatized cysteine residue in the N-terminal (LAAMEDEPSNDN-C), had a theoretical relative molecular mass of 1408.48 D with a measured relative molecular mass of 1407.90 D and a purity exceeding 85.5%. The titer of the antiserum was more than 1:512 000, and the purified IgG antibody concentration was 0.965 mg/ml after purification with Protein A. At a 1:1000 dilution (final concentration of 1μg/ml), the titer of the purified IgG antibody was more than 1: 256 000. The purified IgG antibody could be used at 1:100 to 1:200 dilutions for for immunocytological examination of formalin-fixed cells. Conclusion The anti-NKAα2 EM1 polyclonal antibodies obtained can be used in ELISA and immunocytochemistry for detecting the sodium pump alpha 2 subunit in formalin-fixed tissue or cells to facilitate investigation of the relationship between sodium pump and hypertension.