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The Structural Characterization of Recombinant Bovine Somatotropin Expressed in Escherichia coli (재조합 소성장호르몬의 구조적 특성)

  • 김정호;김훈주박은숙김준
    • KSBB Journal
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    • v.9 no.2
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    • pp.165-173
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    • 1994

  • In this paper we have described the structural characterization of recombinant bovine somatotropin produced in Escherichia coli. Recombinant bovine somatotropin consists of 191 amino acid residues with a calculated molecular weight of 21,802 Da. For fragmentation of recombinant bovine somatotropin, we have used trypsin, Staphylococcus aureus V8 pretease, CNBr, and mild acid hydrolysis method. Digestion and cleavage with these proteases and chemicals yielded peptides of various size for amino acid sequence determination. The N-terminal sequence analysis was carried out up to thirty residues. Because the design of the recombinant bovine somatotropin gene for expression was such that the coding sequence begins with an initiation codon, AUG, before Ala, the first amino acid of bovine somatotropin, we could expect the initial amino acid as N-formyl Met. But the first amino acid of this protein, expressed in E. coli cells as inclusion bodies, was Ala. And the amino acid composition of RP-HPLC purified recombinant bovine somatotropin was determined and no essencial difference was observed. The amino acid sequence of the recombinant bovine somatotropin was identical to that predicted from its recombinant gene. There was no processing or replacement of amino acid residues in recombinant bovine somatotropin expressed in E. coli. The hydropathy plot of recombinant bovine somatotropin revealed a hydrophobic region at the NH2-terminus and hydrophilic region at the COOH-terminus. The E. coli expression system is thought to be valuable for the expression of recombinant bovine somatotropin because protein was processed to remove the N-terminal Met residue by methionyl-aminopeptidase autonomously.

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Development of Variation Marker of Myzus persicae by Altitude (고도에 따른 지역별 복숭아혹진딧물 집단 변이 마커 개발)

  • Kim, Ju-Il;Kwon, Min
    • Korean journal of applied entomology
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    • v.50 no.4
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    • pp.325-333
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    • 2011

  • This study focused on the green peach aphid, Myzus persicae, as an indicator pest in Chinese cabbage cultivation to develop a genetic marker. We hypothesized that M. persicae gene flow is related to climate change. Genetic variation was analyzed using five local populations collected at different altitudes (157 m, 296 m, 560 m, 756 m and 932 m above sea level) in Hoengseong, Pyeongchang, and Gangneung areas, plus a laboratory strain used as an outgroup. There were no differences in ecological characteristics among strains. Esterase isozyme pattern and inter-simple sequence repeat (ISSR) PCR results showed significantly different bands between laboratory and wild, local populations. However, there was no difference among local populations. Partial fragments of ribosomal RNA (rRNA) and mitochondrial cytochrome oxidase I (mtCO I) were amplified and their nucleotide sequence was analyzed. Single nucleotide polymorphisms (SNPs) were detected in internal transcribed spacer-2 (ITS-2) and mtCO I regions among the five local populations. These SNPs can be use to discriminate different populations of M. persicae to monitor gene flow.

  • https://doi.org/10.5656/KSAE.2011.11.0.59 인용 PDF KSCI

Cloning and expression of lin-28 homolog B gene in the onset of puberty in Duolang sheep

  • Xing, Feng;Zhang, Chaoyang;Kong, Zhengquan
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.1
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    • pp.23-30
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    • 2019

  • Objective: Recent studies have demonstrated that lin-28 homolog B (LIN28B)/miRNA let-7 (let-7) plays a role in the regulation of pubertal onset in mammals. However, the role of LIN28B/let-7 in the onset of ovine puberty remains unknown. We cloned the Duolang sheep Lin28B cDNA sequence, detected the expression change of LIN28B, let-7a and let-7g in hypothalamus, pituitary and ovary tissues at three different pubertal stages. Methods: The reverse transcriptase polymerase chain reaction (RT-PCR) was used to clone the cDNA sequence of LIN28B gene from Duolang sheep and the bioinformatics methods were applied to analyze the amino acid sequence of LIN28B protein. The mRNA expression levels of the LIN28B gene at different pubertal stages were examined by real time RT-PCR. Results: LIN28B cDNA of Duolang sheep was cloned, and two transcripts were obtained. The amino acid sequence of transcript 1 shares 99.60%, 98.78%, and 94.80% identity with those of goat, wild yak and pig, respectively. Strong LIN28B mRNA expression was detected in the hypothalamus, pituitary, ovary, oviduct and uterus, while moderate expression was found in the liver, kidney, spleen and heart, weak expression was observed in the heart. No expression was found in the lungs. Quantitative real-time PCR (QPCR) and western-blot analysis revealed that the LIN28B was highly expressed in the hypothalamus and ovary at prepuberty stages, and this expression significantly decreased from the prepuberty to puberty stages (p<0.05). Markedly increased levels of mRNA expression were detected in the pituitary from prepuberty to puberty (p<0.05) and then significantly decreased from puberty to post-puberty (p<0.05). The expression levels of let-7a and let-7g showed no significant changes among different pubertal stages (p>0.05). Conclusion: These results provided a foundation for determining the functions of LIN28B/let-7 and their role in the onset of sheep puberty.

  • https://doi.org/10.5713/ajas.18.0276 인용 PDF KSCI