Study On Cloning Of Gal-6 Gene Fragment And Expression Of Mature Peptide Gene In Poultry

Beta-defensin,an antimicrobial peptide in poultry,showes activity of antimicrobial and function of enhances immunity and little is known about resistivity for pathogenic microrganism,it is one of the focus for many scholar.To investigate the distribution expression information and antimicrobial activity in poultry,the study mainly applied molecular biology and molecular cloning technology.The fragment of Gal-6 was cloned from the tissues of poultry(Three Yellow broile,Wan Xi White goose and Chao Hu duck).The cloning vector of pGEM-T Easy-Gal-6 and expression vector of pGEX-4T-1-Gal-6 were constructed,the primary biologic activity of purified product was investigated.The results were showed as followed:1.Trizol Reagent was adopted to extract the RNA in the marrow of Three Yellow Broiler, Wan Xi White goose and sixteen different tissues of Chao Hu duck.28s and 18s RNA bands were found by 1.0%agarose gel electrophoresis,The OD values of RNA at wavelenghs of 260nm and 280nm were determinated by spectrophotometer,The ratios of OD₂₆₀/OD₂₈₀ were between 1.8 and 2.0.The results suggested that the purity of obtained RNA was high enough to satisfy the requirement of experiment.2.According to the reported cDNA gene sequence(NM₀01001193) of Gallinacin beta-defensin,two pairs of primers were designed,P₁/P₂ amplified Gal-6 gene and P₃/P₄ amplified the gene of mature peptide.RNA from the tissues was reverse transcribed into a first-strand cDNA with random primers.The objective gene fragment were cloned successfully from the tissues of Three Yellow broiler,Wan Xi White goose and Chao Hu duck,by reverse transcription-polymerase chain reaction(RT-PCR),then the produces purified and recovered, then the cloning vector of pGEM-T Easy-Gal-6 was constructed.The recombinant plasmid that was extracted and sequenced was compared by BLAST of www.ncbi.com.Compared with gene fragments registered in GenBank,there was a difference in the 246^th bp of the sequenced 249 bases of Three Yellow Broiler,T mutated C,the highest degree of identity in nucleotide was 99%;the gene sequence of Wan Xi White goose was completely homology,the highest degree of identity in nucleotide was 100%;two bases were lack in the sequenced fragment of ChaoHu duck,only 247bp,there were lack of T in the 12^th bp and 242^th respectively,the highest degree of identity in nucleotide was 99%;The cDNA fragment coded 67 amino acid residues,comprised of signal peptide with amino acid residues,propriece peptide with 5 amino acid residues and mature peptide with 42 amino acid residues.The mature peptide sequences,126bp,were amplified by nested PCR from a pair of primer of P₃/P₄ and the mould of recombinant plasmid pGEM-T Easy-Gal-6.3.To detect Gal-6 distribution information of Chao Hu duck in the sixteen tissues,including intestine,kidney and liver.The results showed that object gene fragments amplified by PCR, were not in kidney,skin and bursa of fabricius,other 13 samples was positive.4.The gene sequences Gal-6 of duck and goose were submitted to GenBank,the GenBank accession number were EU366148 and EU606039,respectively.It is the first reported in GenBank.5.The expression vector was constructed with mature peptide sequence of Gal-6 gene in the tissues of Chao Hu duck.The mature peptide sequence,which was purified and double enzyme cured,was inserted into the gene point of plasmid pGEX-4T-1- Gal-6 vector which was also double enzyme cured,then positive recombinant plasmid was identified by PCR.The amplified positive recombinant plasmid was optimized for induce with IPTG expression of time and temperature,following SDS-PAGE detected the expression information.The result were showed:the fusion protein of GST-Gal-6 was found in the point of 33kD,which meant the expression vector was constructed successfully,it was the best to induce expression with IPTG for four hours,there were fusion protein in the supematant and sediment at 30℃and 37℃,but there were more soluble fusion protein in the supernatant at 30℃.The supernatant product was purified by Glutathione Sepharose chelation affinity chromatography and upper purified GST-Gal-6 fusion protein was received.The analysis of spectrophotometer revealed that expression level of GST-Gal6 was 0.2613mg/mL.The biological activity of expression production in vitro was detected by monlayer agar plate diffusion method it appeared activity of antimicrobial.The above results showed:the objective gene fragments were cloned from the tissues of laboratory animals and constructed the cloning vector and expression vector of mature peptide successfully.The objective fusion protein was induced with IPTG,the bacteriostasis study revealed that purified fusion protein had antibacterial,hese results provides scientific guidance for development and application of antibiotic of defensin in animal husbandry.