豬生長激素基因的選殖

Abstract

我們使用guanidine isothiocyanate和熱酚抽取法，從豬腦下垂體中，將全部的RNA抽出。這些RNA在通過oligo d(T)- cellulose column後，收集poly(A)+—RNA部份，並用酒精將RNA沈澱。 將這些RNA經由兔子網狀血球溶出物的細胞外轉譯系統(rabbit reticulocyte lysate in vitro translation system)後，加入老鼠的生長激素注射入猴子所誘導的抗血清和金黃葡萄球菌的蛋白質A做免疫沈澱法(immunoprecipitation)。由12.5％的SDS-PAGE膠電泳圖，我們可以看到分子量在24,000－25,000道耳吞的蛋白質，應該就是豬生長激素的先驅物(precursor)。 我們也用豬腦下垂體抽出的po1y(A)+－RNA為模版，利用傳統的Sl核酸?法和RNaseH法，來合成雙股互補DNA (ds-cDNA)。雙股互補DNA在d(C）tailing後，和d(G) tailing於PstI site的pBR 322黏合(Annealing)，再殖入大腸桿菌K-12 RRI。 這些殖入雙股互補DNA的大腸桿菌K-12 RRI培養在tetracycline的培養皿上，再轉印到Nitrocellulose membrane，然後用SDS和NaOH，將這些大腸桿菌崩解，且將DNA藉著vaccum baking固定在nitrocellulose membrane 上。 由Nick translation將人類生長激素的互補DNA用〔α-22P〕dCTP標示，合成出放射性強度為1×10^8 cpm/μg 的探測子(probe)，做菌株混成實驗(colony hybridization)來驗證重組的互補DNA。在2000個菌株中，有5個菌株可以和探測子有強烈的混成。這5個陽性反應菌株，用鹼性水解法做小量篩選(mini-screen)。抽出的質體DNA用 PstI消化後，透過洋菜膠電泳將DNA片段分開，再轉移到Nitrocellulose membrane上，再和探測子混成。結果有兩段PstI DNA片段，大約500和400個base pairs呈陽性。這些陽性的DNA片段正在分離製備，且準備將DNA定序。

Total RNA was extracted from the porcine pituitary gland with guanidine isothiocyanate and hot phenol method. The RNA was applied to oligo d(T)-cellulose affinity collum, the poly(A)+-RNA fractions were precipitated and pooled. We have characterized the poly(A)+-RNA by in vitro translation using rabbit reticulocyte lysate system, porcine growth hormone precursor has been immunoprecipitated with monkey against rat growth hormone antiserum and Staphylococcus aureus protein A. In 12.5 % SDS-PAGE, a protein band with molecular weight about 24-25K dalton was found, which should be the porcine growth hormone precursor. Porcine pituitary poly(A)+-RNA was used as a template to synthesize the double-stranded complementary DNA. We used traditional S1 nuclease method and RNase H method. The resulting double-stranded cDNA was inserted into the PstI site of pBR322 with the d(C).d(G) tailing technique and subsequently used to transform E.coli K12 RRI. The transformed E.coli RRI clones were grown on tetracycline plates. Colonies were transferred to nitrocellulose filter and lysed with SDS and NaOH method and then fixed by vaccum backing. E.coli clones containing recombinant cDNA were identified by colony hybridization to human growth hormone cDNA probe labled with [α-32P]dCTP by nick translation (specific activity: 1×10^8 cpm/μg). Five out of 2000 clones hybridized strongly with the probe. These positive clones were subsequently mini-screened with alkaline lysis method. The plasmid DNA was digested with PstI, seperated by agarose gel electrophoresis, transferred to nitrocellulose membrane and hybridized with the probe again. Two PstI fragments about 500 and 400 base pairs were positive. The positive fragments were prepared and being sequensed.