豬源補體蛋白C1q的純化及定性與定量之研究

Abstract

本論文以低離子強度溶液來透析沉澱豬源補體蛋白C1q,所得到的產率 為4%,而其specific activity可提升為原來的1696倍,purification fold 則為1700倍.從HPLC系統的分析圖中發現:豬源補體蛋白C1q在native form 或denature form時的peak之shift情況皆與human C1q相類似;從SDS-PAGE 的結果而言,豬源補體蛋白C1q的A, B及C chain的分子量約為29kD,27 kD,24kD;在等電點的分析中發現其pI值約為7.6-8.4附近;在活性的分析中 發現:所純化得的補體蛋白C1q具有溶血活性及能特異的與免疫複體形成很 高的親合性鍵結. The C1q were purified from swine serum by dialysis three times, finally was concentrated by ultrafiltration technique. In all purified stages the fractions with swine C1q were used to mix with sensitized sheep red blood cells to induce erythrocyte hemolysis.In addition, we used uv/visble spectrophotometer to quantitate theswine C1q bioactivity.The results indicated that the specific bioactivityincreased to 1696 fold, the purity reached 95% and from 100 ml swine serum,10 ug C1q was recoveried. In this study, the molecular weight of A, B andC chain of swine C1q, were 29kD, 27kD and 24kD, respectively.In HPLC systemanalysis, the swine C1q's peak pattern was almost the same when compare to that of human C1q in either native form or reducing form. The pI value of swine C1q was 7.6-8.4. We also developed a microplate ELISA method to quantitate swine C1q.The binding ability of swine C1q was also measuredin this study. It is clearly that it can bind strongly with immune complex,but it can bind weakly with free immunoglobulin and antigen.