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huojinlong8610金虫 (小有名气)
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本研究旨在获得版纳微型猪近交系（BMI）生长激素基因编码区序列，揭示其原核表达规律。从版纳微型猪近交系猪脑垂体中提取RNA，采用RT-PCR方法扩增生长激素基因cDNA序列，克隆至pMD18-T载体测序并进行了生物信息学分析。将GH成熟肽区编码序列定向连入表达载体pET-32α(+)，转化入大肠杆菌Rosseta (DE3)感受态细胞中，利用IPTG诱导表达蛋白，通过SDS-PAGE和Western Blot检测并验证了结果。扩增的GH cDNA序列已提交到GenBank，登录号为JQ177096，序列长690 bp，其中CDS长651 bp，编码216个氨基酸，分子量约为24.4 ku，前26个氨基酸残基为信号肽区，成熟肽分子量约为21.7 ku，扩增的GH成熟肽区编码序列长617 bp。GH氨基酸序列比对结果表明其在各猪种中高度保守，BMI的GH氨基酸序列与五指山猪、宁乡猪的相似性为100%，与藏猪、香猪、大乌猪、太湖猪、成华猪、内江猪、荣昌猪、长白、约克夏的相似性为99%，与雅南猪、杜洛克的相似性均为98%。SDS-PAGE凝胶电泳分析得出融合蛋白以包涵体形式存在并高效表达，Western Blot分析证实融合蛋白分子量约为40.6 ku，与预期大小一致。以上结果为进一步探究GH基因对BMI矮小性生长的影响奠定了基础。 This experiment was conducted to obtain the coding sequence of Banna Mini-pig Inbred Line growth hormone (GH) gene and to study its prokaryotic expression. The full-length cDNA of GH gene was cloned from pituitary by RT-PCR and inserted into pMD 18-T vector for sequencing and bioinformatics analysis. Subcloned the GH encoding mature peptide sequence into the prokaryotic expressing vector pET-32α (+) directionally to constitute the recombinant plasmid pET-32α-GH. Then transformed it into the E.coli Rosseta expression bacteria and induced by IPTG in different concentration and time. The recombinant expression products were detected by SDS-PAGE and confirmed by Western Blot analysis. PCR product of GH gene contained 690 nucletides (GenBank No. JQ177096) including a 651 bp CDS. The full-length CDS encoded 216 amino acids with a peried of 26 amino acids of single peptide. The molecular weight of the precursor peptide was 24.4 ku and the mature peptide was 21.7 ku. Coding mature peptide sequence was comprised of 617 nucletides. It was found that the GH amino acids sequence shared 100%, 100%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 98% and 98% identity with those of WZS, Ningxiang pig, Tibetan pig, Xiang pig, Dawu pig, Taihu pig, Chenghua pig, Neijiang pig, Rongchang pig, Landrace, Yorkshire, Yanan pig and Duroc, respectively. SDS-PAGE results showed that the fusion protein expressed in form of inclusion body. The optimal induction condition is 0.01 mmol•L-1 IPTG inducted for 4 h. Western blot analysis indicated that the recombinant protein molecular weight was approximate 40.6 ku and can be recognized by antiserum specifically. Collectively these results will lay the foundation for further studies of the GH effects on the dwarfism of Banna Mini-pig Inbred Line. |
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铭风mf
金虫 (正式写手)
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huojinlong8610: 金币+60, 翻译EPI+1, ★有帮助 2012-06-28 16:12:27
huojinlong8610: 金币+60, 翻译EPI+1, ★有帮助 2012-06-28 16:12:27
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删除的单词全部用大写字母表示；添加或修改的用括号括了起来，good luck THIS EXPERIMENT (The research) was conducted to obtain the coding sequence of Banna Mini-pig Inbred Line growth hormone (GH) gene and to STUDY (identify) its prokaryotic expression. The full-length cDNA of GH gene was cloned from pituitary by RT-PCR and inserted into pMD 18-T vector for sequencing and bioinformatics analysis. SUBCLONED (The) GH encoding mature peptide sequence (was subcloned) into the prokaryotic expressing vector pET-32α (+) directionally to constitute the recombinant plasmid pET-32α-GH. Then it (was transformed) into the E.coli Rosseta expression bacteria and induced by IPTG in different concentration and time. The recombinant expression products were detected by SDS-PAGE and confirmed by Western Blot analysis. PCR product of GH gene (contains) 690 nucletides (GenBank No. JQ177096) including a 651 bp CDS. The full-length CDS encoded 216 amino acids with a (period) of 26 amino acids of single peptide. The molecular weight of the precursor peptide was 24.4 ku and the mature peptide was 21.7 ku. Coding mature peptide sequence was comprised of 617 nucletides. It was found that the GH amino acids sequence shared 100%, 100%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 99%, 98% and 98% identity with those of WZS, Ningxiang pig, Tibetan pig, Xiang pig, Dawu pig, Taihu pig, Chenghua pig, Neijiang pig, Rongchang pig, Landrace, Yorkshire, Yanan pig, (and) Duroc, respectively. SDS-PAGE results showed that the fusion protein expressed in form of inclusion body. The optimal induction condition is 0.01 mmol•L-1 IPTG inducted for 4 (hours). Western blot analysis indicated that the recombinant protein molecular weight was (approximately) 40.6 ku and can be recognized by antiserum specifically. Collectively these results will lay the foundation for further studies of the GH effects on the dwarfism of Banna Mini-pig Inbred Line. |
2楼2012-06-22 19:37:23