重組豬乳鐵蛋白之表現與純化

Abstract

摘要 乳鐵蛋白 (lactoferrin) 是一種人類及動物體內普遍存在的攜鐵醣蛋白，除了可以抑制細菌的生長外，還可促進淋巴細胞的分化，及調節巨嗜細胞 (marcophage)、顆粒細胞 (granulocyte) 的增生，更有抑制癌細胞增生及腫瘤轉移的功能。乳鐵蛋白經胃蛋白□ (pepsin) 消化處理後，會釋放出一功能胜□ (lactoferricin)，具有著比乳鐵蛋白更強的殺菌能力。 我們實驗室嘗試著建立豬乳鐵蛋白及乳鐵蛋白功能胜□的原核細胞表現系統：首先將不同豬乳鐵蛋白基因片段選殖到pET expression vector，在大腸桿菌BL21 (DE3) 進行表現。大量的表現結果造成豬乳鐵蛋白以包涵體 (inclusion body) 的形式堆積在細菌體內，在嘗試著降低誘導溫度與誘導物質濃度後，仍然形成包涵體，接著我們以尿素使包涵體變性後再以梯度透析復性，但仍無法得到可溶具活性的豬乳鐵蛋白。在乳鐵蛋白功能胜□表現方面，我們參考了pET31b眾列胜□表現系統，採用化學合成寡聚核甘酸及聚合□鏈鎖反應兩種選殖的方式，利用大腸桿菌量產人類及豬的乳鐵蛋白功能胜□，經西方轉漬免疫檢測，確定有微量表現，推測可能因此段功能胜□表現後，會對菌體造成毒性，而被抑制其表現。雖然，我們無法順利得到乳鐵蛋白功能胜□，但這個表現系統應可以應用至其他胜□來達到量產的目的。

Abstract Lactoferrin (LF) is an iron-binding glycoprotein that widely exists in human and mammalian body. In certain concentrations LF has a broad spectrum of antimicrobial properties. LF is also involved in the differentiation process of granulocytes and proliferation of macrophages. Moreover, it has been reported that LF exhibited an inhibitory effect on cancer growth and tumor metastasis. When digested by pepsin, LF can release a functional peptide lactoferricin (LFicin), which has a higher antimicrobial activity than LF. We have tried to establish a bacterial expression system for both porcine LF and LFicin. The several parts of coding region of the pLF cDNA was cloned to pET plasmids respectively, then expressed in E. coli BL21 (DE3). However, these recombinant pLFs synthesized in BL21 (DE3) were found to accumulate in the bacteria to form inclusion bodies. This problem could not be solved by decreasing the culture temperature with lower concentration of the inducer, or by the urea denaturation followed by renaturation with gradient dialysis. For the expression of tandem repeats of porcine and human Lficins in E. coli, we have cloned the DNA fragments that obtained via PCR and chemical oligonucleotides synthesis into pET31b. We later confirmed that Lficin had been expressed slightly by the western blotting analysis. The low level expression of the tandem repeats of Lficins is likely due to the toxicity to the bacteria themselves. Although we could not get the functional Lficins, this expression system may be applied to produce other nontoxic peptide successfully.