| 摘要: |
| 【目的】克隆西农萨能羊凝乳酶原基因并进行原核表达，对表达产物凝乳酶原复性后进行活性检测，为西农萨能羊凝乳酶制剂的微生物发酵生产奠定基础。【方法】从西北农林科技大学西农萨能羊原种场新生公羔羊的皱胃组织中提取总RNA，通过RT-PCR方法获得西农萨能羊凝乳酶原前体的编码序列,纯化后与pMD-18T载体连接，转化大肠杆菌JM109，通过双酶切和序列测定，获得了凝乳酶基因全长编码区序列。将该基因连接到原核表达载体pET-30a中，构建重组菌pET-30a/Chymosin，经酶切、测序鉴定后进行凝乳酶的小量表达、大量表达、Western杂交鉴定、纯化、复性和活性测定。【结果】通过RT-PCR方法获得了西农萨能羊凝乳酶原前体的编码序列，并构建了重组菌pET-30a/Chymosin；通过体外原核表达获得了西农萨能羊凝乳酶原，将酶原进行复性后测得重组菌酶活力为93.2 U/mL。【结论】利用西农萨能羊凝乳酶原基因，通过构建原核表达载体和体外表达可以得到凝乳酶原，通过蛋白纯化和复性可得到有活性的凝乳酶。 |
| 关键词: 西农萨能羊 凝乳酶 原核表达载体 |
| DOI： |
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| 基金项目:河南省科技攻关计划项目（0624030009） |
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| Cloning and prokaryotic expression of preprochymosin cDNA of Xinong Saanen Goat |
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| Abstract: |
| 【Objective】The research was done to clone and prokaryotic express the preprochymosin cDNA of Xinong Saanen Goat,and test the activity of recombinant protein after renaturation.【Method】Preprochymosin cDNA was obtained from total RNA isolated from the abomasum of Xinong Saanen Goat by RT-PCR method.The purified RT-PCR procucts and pMD-18T vector were ligated and transformed into host strain E.coli JM109.The full length of Chymosin gene and pET-30a vector were ligated and the recombinant plasmid pET-30a/Chymosin was constructed successfully.The recombinant was identificatied,expressed in vitro,then purificated and the activity performed.【Result】 Preprochymosin cDNA was obtained by RT-PCR method and the recombinant plasmid pET-30a/Chymosin was contructed.Recombinant protein preprochymosin of Xinong Saanen was obtained by Prokaryotic expression in vitro,the enzyme activity tested after renaturation was 93.2 U/mL.【Conclusion】 Preprochymosin gene can be obtained by constructing recombinant plasmid and expressing in vitro.Activity tests showed that the enzyme which was obtained according to purification and renaturation of preprochymosin had milk-clotting activities. |
| Key words: Xinong Saanen Goat chymosin prokaryotic expression vector |
