鯉魚性腺刺激素β次單元基因在大腸桿菌之表現及產物特性之研究

Abstract

本實驗利用以前選殖成功，含鯉魚腦下腺β次單元基因之 cDNA clone一pCP916 ，應用多鏈聚合?反應（polymerase chain reaction , PCR）方法，取得此段基因後，將其送入表現載體（expression vector）-pGEX-2T ．經 IPTG ( isopropyl-β-thiogalactopyranoside) induction 可得一fusion protein。 由於從鯉魚腦下腺所得之性腺刺激素β次單元，較從 cDNA 所得之胺基酸序列，在 C 端少兩個胺基酸．因而我們設計兩組prime去做PCR ．結果所得，檢視其 DNA sequence ，可取得所要的 pro-form（β1）及 mature form（β2）兩種次單元基因．並且於pGEX－2T 中，成功的表現出來．在通過 glutathione -agarose affinity column後，經 thrombin digestion 也可淤Western blot 中觀察到位於14kDa之β次單元。 其次拿 fusion protein 對guinea pig做免疫反應，取得之antibody，不僅對原來之fusion protein有反應，同時對鯉魚腦下腺之crude extract，亦可偵測出帶醣β次單元的single band ．所以此fusion protein不僅攜帶了所預期的β次單元，於epitope上與帶醣之nativeβ次單元仍有極高相似性。 綜合實驗所得，利用此一表現系統，可得不帶醣simple protein，供進一步研究用．然而在量的方面，也就是fusion protein 的產量，及經thrombin digestion 後所得之β次單元之量上，在本實驗中，並非理想，這點則尚須改善。

In this study, we use E.coli expression system to produce two fusion proteins each carrying one of the two β subunits of carp gonadotropin (GTHβ) on the carboxyl terminus. There are two forms of GTHβ (β1&β2). Deduced from cDNA sequence, the β1 is a pro-form with 117 amino acid residues, and the β2 is a mature form without the last two amino acid residues on its carboxyl terminus. Two sets of primers were designed and synthesized for amplification of the DNA fragments encoding these two forms of GTHβ by polymerase chain reaction (PCR), and then the DNA fragments were subcloned to the expression vector, pGEX-2T, respectively. Both of the two GTHβ subunits were successfully expressed as fusion proteins, and detected by Western blot using an anti-serum against the native GTHβ which was purified from carp pituitary glands. Also, both of the two crude extracts were further purified by being passed an affinity column, glutathione-agarose affinity column, and then the purified fusion protein were digested with thrombin o remove the carrier protein, the glutathione S-transferase. In addition, the purified fusion proteins were injected into guinea pigs, respectively, to induce anti-β1and -β2 sera. Both antisera obtained could cross-react with either pro-form (β1) or mature form (β2) a well and could be also used to detect a single band of GTHβ from the crude extract of carp pituitary glands in Western blot.