郑州大学学报（医学版）
鄭州大學學報（醫學版）
정주대학학보（의학판）
JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES)
2013年
5期
581-583
,共3页
李一帆%崔柳青%韩康%薛乐勋
李一帆%崔柳青%韓康%薛樂勛
리일범%최류청%한강%설악훈
canstatin%翻译增强序列%叶绿体%杜氏盐藻
canstatin%翻譯增彊序列%葉綠體%杜氏鹽藻
canstatin%번역증강서렬%협록체%두씨염조
canstatin%translation enhancer sequence%chloroplast%Dunaliella salina
目的：构建含翻译增强序列的人canstatin杜氏盐藻叶绿体重组表达载体。方法：采用RTP-CR 的方法，获得含有人canstatin 的cDNA片段以及分别在5’UTR下游和3’UTR上游添加了翻译增强序列（ UUAACUUUA）的人canstatin cDNA 片段，将得到的目的片段分别连接到PMD18-T载体上，利用该载体将目的片段连接到荧光素酶基因Lux Ct表达盒中并进行酶切鉴定。结果：RT-PCR分别得到约700 bp的cDNA片段并克隆到PMD18-T载体上。用PaeR7Ⅰ、SphⅠ双酶切 Lux Ct质粒，得到2100 bp和10000 bp 的2条片段。然后，将大片段回收后分别与上一步得到的cDNA片段进行连接，酶切鉴定结果显示含有翻译增强序列的人canstatin 片段成功插入到Lux Ct质粒中。结论：成功构建了含有翻译增强序列的人canstatin杜氏盐藻叶绿体重组表达载体。
目的：構建含翻譯增彊序列的人canstatin杜氏鹽藻葉綠體重組錶達載體。方法：採用RTP-CR 的方法，穫得含有人canstatin 的cDNA片段以及分彆在5’UTR下遊和3’UTR上遊添加瞭翻譯增彊序列（ UUAACUUUA）的人canstatin cDNA 片段，將得到的目的片段分彆連接到PMD18-T載體上，利用該載體將目的片段連接到熒光素酶基因Lux Ct錶達盒中併進行酶切鑒定。結果：RT-PCR分彆得到約700 bp的cDNA片段併剋隆到PMD18-T載體上。用PaeR7Ⅰ、SphⅠ雙酶切 Lux Ct質粒，得到2100 bp和10000 bp 的2條片段。然後，將大片段迴收後分彆與上一步得到的cDNA片段進行連接，酶切鑒定結果顯示含有翻譯增彊序列的人canstatin 片段成功插入到Lux Ct質粒中。結論：成功構建瞭含有翻譯增彊序列的人canstatin杜氏鹽藻葉綠體重組錶達載體。
목적：구건함번역증강서렬적인canstatin두씨염조협록체중조표체재체。방법：채용RTP-CR 적방법，획득함유인canstatin 적cDNA편단이급분별재5’UTR하유화3’UTR상유첨가료번역증강서렬（ UUAACUUUA）적인canstatin cDNA 편단，장득도적목적편단분별련접도PMD18-T재체상，이용해재체장목적편단련접도형광소매기인Lux Ct표체합중병진행매절감정。결과：RT-PCR분별득도약700 bp적cDNA편단병극륭도PMD18-T재체상。용PaeR7Ⅰ、SphⅠ쌍매절 Lux Ct질립，득도2100 bp화10000 bp 적2조편단。연후，장대편단회수후분별여상일보득도적cDNA편단진행련접，매절감정결과현시함유번역증강서렬적인canstatin 편단성공삽입도Lux Ct질립중。결론：성공구건료함유번역증강서렬적인canstatin두씨염조협록체중조표체재체。
Aim: To construct a chloroplast expression vector of Dunaliella salina for recombinant human canstatin with translation enhancer sequence .Methods:The cDNA fragments encoding the human canstatin , the human canstatin with translation enhancer sequence ( UUAACUUUA) in downstream of 5'UTR and that with translation enhancer sequence (UUAACUUUA) in upstream of 3'UTR were obtained by RT-PCR, respectively.Subsequently, the fragments were cloned into the PMD18-T vector and were identified by digestion of restriction enzymes , respectively .The correct fragments were subcloned into the expression cassette in Lux Ct ,and then the recombinant plasmid was identified by digestion of restriction enzymes.Results:The RT-PCR products of about 700 bp were cloned into the PMD18-T vector.Lux Ct plasmid digested by PaeR7Ⅰand SphⅠproduced two fragments , one was of 2 100 bp and the other was of 10 000 bp, and they were recy-cled and ligated with the cDNA fragments of canstatin .The results of enzyme digestion showed that canstatin fragments with translation enhancer sequence were correctly inserted into the Lux Ct plamid .Conclusion:The chloroplast expression vec-tor of Dunaliella salina for recombinant human canstatin with translation enhancer sequence has been constructed success -fully.
