鯉魚頭腎膽固醇側鏈裂?之cDNA選殖與核酸定序

Abstract

本實驗以基因選殖的方法，研究鯉魚頭腎瞻固醇側進解裂(Cholesterol side chain cleavage enzyme, P450 scc) 的初級結構：先以Guanidium thiocyanate-CsCl方法製配頭腎RNA，以Oligo-dT cellulose column 篩選含 poly-A的mRNA，再以Reverse transcriptase裂成 complementary DNA (cDNA) cDNA之兩端各銜接 ECORI linker，之後將之嵌入puc 19之ECOR工限制作用點，並以E. coli JM101為宿主繁殖上述重組的質體，建立魚頭腎cDNA庫： 本實驗以人類P450 scc的cDNA為探針，篩選上述鯉魚頭腎cDNA庫中P450 scc的scc cDNA。篩選時，先以菌落雜交（Colony Hybrdization）行之，選取疑似的菌落，再製備質體DNA，以ECORI處理，進行電泳解析，並於電泳繆以上進行分子雜交，確認合有P450 scc之cDNA質體，以此方法計得二個含P450 scc之cDNA質體： 木實驗僅就其中之一的。cDNA加以探討，為欲達成完整的定序工作，本實驗以Pst I及Ava I將P450 scc的cDNA分成5個段落再分別選殖，之後分別以Sanyer's的Dideoxy nucleotide方法加以定序，之後銜接各段落，得到近乎完整的核?酸序列。 鯉魚頭腎P450scc cDNA的全長，由電泳估計，約為1.8Kb，目前的定序工作尚‘餘3’非轉譯區，3'untranslated region)未完成，由已完成的核?酸定序工作，得知17bP的5’非轉譯區，從起始密碼(Initiation Codon)至終止密碼(Termination Codon)共有1560 bp，可轉譯為519個氨基酸的P450 scc之前前質(Precursor)。 經由氨基酸序列之相似性比較，鯉魚的P450 scc與人類者有93%之相似性，而與牛者有70%的相似性。

The cDNA library base on mRNA from carp head kidney was constructed. A total amount of 2064 clones were obtained. For screening of cDNA encoding cholesterol side chain cleavage enzyme (P450 scc) of carp head kidney, the cDNA encoding human P450 scc was nick translated and used as probe. There were positive clones, identified by colony hybridization and in situ gel hybridization, obtained in the present study, a clone, denoted as CHK-7 was studied. The cDMA was estimated to be about 1.8 kb by agarose electrophoresis. For sequencing, CHK-7 was further subcloned. The present study almost finished the complete sequence of the cDNA except about 200 bp of 3' untranslate regions. Among the 1624 bp thus sequenced, it consisted of 50 bp 5’ untranslated regions, 1557 bp of open reading frame. The open reading frame started from the initiation radon. AT and terminated at the termination codon, TAA. a total of 519 amino acid residues were translated. In comparison with the amino acid sequences deduced from the cDNAs encoding human and bovine P450 scc, it was found there were 93% and 70% homology between fish vs human and fish vs bovine P450 scc, respectively. Furtherm-ore the heme binding domain of P450 scc among those three species were highly conserved.