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Modification、Cloning and Prokaryotic Expression of the Gene Encoding Antibacterial Peptide PolyphemusinⅡ from Tachypleus Tridentatus

Author: ZhangZuo
Tutor: DanWenLu；HanWenZuo
School: Xinjiang Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Chinese horseshoe crab Antibacterial peptide polyphemusin Ⅱ Clone The prokaryotic expression
CLC: Q789
Type: Master's thesis
Year: 2005
Downloads: 168
Quote: 1
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Abstract


Tachyplesin (tachyplesin) is the general term for antimicrobial peptides in the horseshoe crab blood has broad-spectrum bactericidal, inhibiting viral and anti-tumor effect and, therefore, has aroused extensive attention of scholars at home and abroad. A limited number of natural horseshoe crab, farmed difficult, extracted from horseshoe crab blood cells tachyplesin natural products can not meet the demand, and the use of molecular biology techniques, develop produce tachyplesin engineering strain, is a practical approach has broad application prospects. Therefore, this study classic means of molecular biology will transform the the Limulus antimicrobial peptides polyphemusin Ⅱ coding sequence into E. coli highly expressed in the industrial production of the antibacterial peptide polyphemusin Ⅱ engineered bacteria to make it possible to obtain. The first part of this study, carried out the Chinese horseshoe crab antimicrobial peptides polyphemusin Ⅱ genetic modification and cloning work. Collecting the blood of the horseshoe crab, Limulus of amebic blood cells total RNA was extracted using Trizol reagent. Horseshoe crab antimicrobial peptides of the Americas polyphemusin Ⅱ amino acid sequence has been published by the NCBI, the original antimicrobial peptide gene primers were designed to transform the Asn codon: Add at the end of the open reading frame, so that the antimicrobial peptide carboxyl-terminal amidation to improve its stability followed by RT-PCR and antibacterial activity of antimicrobial peptides target fragment was transformed rr, then its cloning vector pMD18-T and sequenced positive clones were screened limulus the antibacterial peptide polyphemusin Ⅱ gene. The second part of the prokaryotic expression vector construction, expression and detection. Recombinant cloning plasmid pMD18-T-rr double digested with Nde Ⅰ, EcoR Ⅰ, and after the appropriate restriction enzyme digestion processing prokaryotic expression vector pET-28c () to connect, and transformed into E.coli JM109 competent cells by PCR and sequencing confirmatory positive recombinant plasmid, .. in the host bacteria E. coli BL21 (DE3), induced expression. SDS-PAGE analysis results found that the cleavage product of the expression in bacteria and control bacteria compared to the emergence of a new protein band, and its size is approximately 2.5KD. TLC scanning results show that the expression of antimicrobial peptides protein accounted for 27.21% of the total bacterial proteins. Western blot analysis, about 2.5KD at specific reaction with the expression product with the natural tachyplesin positive serum specifically reacted. Extracted inclusion bodies get the antimicrobial peptides beginning of extracts for antibacterial experiments, the results found that showed significant antibacterial activity in vitro. In summary, this study successfully the antibacterial peptide polyphemusin Ⅱ gene to be modified and transformed into E. coli expression, access to the highly efficient antibacterial activity of the expression product, which laid the foundation for genetic engineering research tachyplesin its industrial development and application.

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