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北京石油化工學(xué)院2026年研究生招生接收調(diào)劑公告

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本研究旨在獲得版納微型豬近交系（BMI）生長(zhǎng)激素基因編碼區(qū)序列，揭示其原核表達(dá)規(guī)律。從版納微型豬近交系豬腦垂體中提取RNA，采用RT-PCR方法擴(kuò)增生長(zhǎng)激素基因cDNA序列，克隆至pMD18-T載體測(cè)序并進(jìn)行了生物信息學(xué)分析。將GH成熟肽區(qū)編碼序列定向連入表達(dá)載體pET-32α(+)，轉(zhuǎn)化入大腸桿菌Rosseta (DE3)感受態(tài)細(xì)胞中，利用IPTG誘導(dǎo)表達(dá)蛋白，通過(guò)SDS-PAGE和Western Blot檢測(cè)并驗(yàn)證了結(jié)果。擴(kuò)增的GH cDNA序列已提交到GenBank，登錄號(hào)為JQ177096，序列長(zhǎng)690 bp，其中CDS長(zhǎng)651 bp，編碼216個(gè)氨基酸，分子量約為24.4 ku，前26個(gè)氨基酸殘基為信號(hào)肽區(qū)，成熟肽分子量約為21.7 ku，擴(kuò)增的GH成熟肽區(qū)編碼序列長(zhǎng)617 bp。GH氨基酸序列比對(duì)結(jié)果表明其在各豬種中高度保守，BMI的GH氨基酸序列與五指山豬、寧鄉(xiāng)豬的相似性為100%，與藏豬、香豬、大烏豬、太湖豬、成華豬、內(nèi)江豬、榮昌豬、長(zhǎng)白、約克夏的相似性為99%，與雅南豬、杜洛克的相似性均為98%。SDS-PAGE凝膠電泳分析得出融合蛋白以包涵體形式存在并高效表達(dá)，Western Blot分析證實(shí)融合蛋白分子量約為40.6 ku，與預(yù)期大小一致。以上結(jié)果為進(jìn)一步探究GH基因?qū)MI矮小性生長(zhǎng)的影響奠定了基礎(chǔ)。
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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huojinlong8610: 金幣+60, 翻譯EPI+1, ★有幫助 2012-06-28 16:12:27

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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.

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2樓2012-06-22 19:37:23

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