CN100543138C - The preparation method of reorganization chicken-beta alexin protein Gal-9-9 - Google Patents
The preparation method of reorganization chicken-beta alexin protein Gal-9-9 Download PDFInfo
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- CN100543138C CN100543138C CNB2006101511758A CN200610151175A CN100543138C CN 100543138 C CN100543138 C CN 100543138C CN B2006101511758 A CNB2006101511758 A CN B2006101511758A CN 200610151175 A CN200610151175 A CN 200610151175A CN 100543138 C CN100543138 C CN 100543138C
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Abstract
The present invention is to provide a kind of preparation method of the chicken-beta alexin protein Gal-9 of recombinating-9.The present invention's Protocols in Molecular Biology, from the chicken vivo clone to chicken-beta-defensin Gal-9 gene, select for use suitable expressive host at vivoexpression chicken-beta-defensin Gal-9 albumen.Discover that further chicken-beta-defensin Gal-9 albumen has broad spectrum antibiotic activity and immunologic enhancement.The reorganization chicken Gal-9 albumen that adopts method of the present invention to obtain: 1) can press down bacterium such as killing intestinal bacteria, Salmonellas, streptococcus aureus, 2) anti-avian infectious bronchitis virus, 3) after chicken is oral after 1 week, improve anti-avian infectious bronchitis virus antibody horizontal 10-20% in the immune chicken serum, improve chicken splenic lymphocyte transformation efficiency 40-50%.
Description
One, technical field
That the present invention relates to is a kind of preparation method of biomaterial, specifically a kind of preparation method with biomaterial of broad spectrum antibiotic activity and immunologic enhancement.
Two, background technology
Because feeding antibiotic is widely-used in the feed, causes microbiotic residual in carcass and livestock product, and to causing the resistance of animal, brings very big threat to human health and environment.Therefore press for a kind of natural free of contamination fodder additives and be applied in the feed, promote growth of animal, reduce feeding cost.Reorganization chicken-beta alexin protein (gallinacins) Gal-9 is naturally occurring in the chicken body, contains 67 of amino-acid residues, has the micromolecule polypeptide of broad spectrum antibiotic activity.Body, inside and outside have broad spectrum antibiotic activity and immunologic enhancement, for this invention research and application provide theoretical basis.
Three, summary of the invention
The bright purpose of this law is to provide the preparation method of the reorganization chicken-beta alexin protein Gal-9-9 that has broad spectrum antibiotic activity and immunologic enhancement in.
The object of the present invention is achieved like this:
1. design Auele Specific Primer according to the chicken of having delivered-beta-defensin Gal-9 gene order,
Upstream primer: 5 '-GGA TCC CCG GAA TTC ATG CAG ATC CTG CCT CTC-3 ',
Downstream primer: 5`-TCA GGA ATA CCA TCG GCT CCG GCA GCA GAA-3`;
2. adopt from chicken tongue tissue, increase chicken beta-defensin Gal-9 gene and being cloned on the PMD-T carrier of RT-PCR method, determine that through sequencing its gene order is for (dash area is a target gene, in the square frame is primer, and italic is respectively initiator codon and terminator codon):
TCCTCCTCCTGATGCTCCGGGCAGAACCAGGGCTGTCCCTTGC
TCGAGGATTACCCCAGGACTGTGAGCGCCGTGGGGGCTTCTG
CTCCCACAAGTCATGTCCTCCAGGGATCGGCCGCATTGGCCTC
ATCTCTAAGGATCCCCGGGTACCGAGCTCGAATTCGTAATCAT
GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCCGCTCACAA
TTCCACACAACATACC
3. according to chicken-beta-defensin Gal-9 gene order characteristics, with EcoRI and SalI double enzyme site with Gal-9 gene subclone in prokaryotic expression carrier PGEX-6p-1, make up recombinant expression vector PGEX-6p-1-Gal-9, with its transformed into escherichia coli BL21 competent cell, Amp+ screening positive clone;
4. the single bacterium colony of the e. coli bl21 37 ℃ of shaking culture in the Amp+LB substratum that contain positive recombinant plasmid, add IPTG when treating that the OD value reaches 0.3-0.5, final concentration is that 0.6mM induces, induce and adopt 1ml bacterium sample after 4 hours, and left the heart 5 minutes in 4 ℃, 5000, the results bacterial precipitation, in precipitation, add the long-pending 1 * sds gel sample-loading buffer of 1/10 bacteria liquid, boiled ice bath 2 minutes 5 minutes.
5. reclaim Gal-9 albumen through the affinity chromatography column purification.
Gal-9 fusion rotein behind the abduction delivering carries out the SDS-PAGE Analysis and Identification, gets the sample 15ul/ hole of handling, and carries out SDS-PAGE electrophoresis (spacer gel concentration is 5%, and separation gel is 12%).Behind the electrophoresis, through coomassie brilliant blue staining, methyl alcohol-glacial acetic acid destainer decolouring.By the thin layer gray scale scanning, determine the protein expression amount again.Obtain the specifically expressing band of molecular weight 32KD,, prove the Gal-9 differential protein through western blot analysis.The protein expression amount is 40%.
Through the affinity chromatography column purification reclaim Gal-9 albumen in vivo, measure the proteic antibiotic and immunization of chicken Gal-9 of recombinating outward, discover reorganization chicken Gal-9 albumen: 1) can press down bacterium such as killing intestinal bacteria, Salmonellas, streptococcus aureus, 2) anti-avian infectious bronchitis virus, 3) after chicken is oral after 1 week, improve anti-avian infectious bronchitis virus antibody horizontal 10-20% in the immune chicken serum, improve chicken splenic lymphocyte transformation efficiency 40-50%.
The present invention's Protocols in Molecular Biology, from the chicken vivo clone to chicken-beta-defensin Gal-9 gene, select for use suitable expressive host at vivoexpression chicken-beta-defensin Gal-9 albumen.Discover that further chicken-beta-defensin Gal-9 albumen has broad spectrum antibiotic activity and immunologic enhancement.
Four, description of drawings
Fig. 1 is for adopting the RT-PCR method chicken beta-defensin Gal-9 gene photo that increases from chicken tongue tissue;
Fig. 2 is Gal-9 gene thin layer gray scale scanning figure.
Five, embodiment
For a more detailed description to the utility model for example below in conjunction with accompanying drawing:
1. design Auele Specific Primer according to the chicken of having delivered-beta-defensin Gal-9 gene order,
Upstream primer: 5 '-GGA TCC CCG GAA TTC ATG CAG ATC CTG CCT CTC-3 ',
Downstream primer: 5`-TCA GGA ATA CCA TCG GCT CCG GCA GCA GAA-3`
2. adopt from chicken tongue tissue, increase chicken beta-defensin Gal-9 gene and being cloned on the PMD-T carrier of RT-PCR method, be defined as chicken-beta-defensin Gal-9 gene through sequencing.(dash area is a target gene sequences, and all the other are carrier, is primer in the square frame, and italic is respectively initiator codon and terminator codon):
CTCTTTGCTG
TCCTCCTCCTGATGCTCCGGGCAGAACCAGGGCTGTCCCTTGC
TCGAGGATTACCCCAGGACTGTGAGCGCCGTGGGGGCTTCTG
CTCCCACAAGTCATGTCCTCCAGGGATCGGCCG
TGCTCCAAGGAAGACTTCTGCTGCCGGAGCCGATGGTA
GA
AAGCTTGGGA
ATCTCTAAGGATCCCCGGGTACCGAGCTCGAATTCGTAATCAT
GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCCGCTCACAA
TTCCACACAACATACC
3. construction of prokaryotic expression vector: according to chicken-beta-defensin Gal-9 gene order characteristics, with EcoRI and SalI double enzyme site with Gal-9 gene subclone in prokaryotic expression carrier PGEX-6p-1, make up recombinant expression vector PGEX-6p-1-Gal-9, with its transformed into escherichia coli BL21 competent cell, Amp+ screening positive clone.In a small amount method is extracted plasmid and is carried out enzyme and cut and deliver Shanghai bio-engineering corporation after identifying with PCR and carry out sequencing.
4.Gal-9 the abduction delivering of fusion rotein: the single bacterium colony of the e. coli bl21 37 ℃ of shaking culture in the Amp+LB substratum that contain positive recombinant plasmid.Treat that the OD value reaches at 0.3 ~ 0.5 o'clock and adds IPTG (final concentration is 0.6mM) and induce.Adopt 1ml bacterium sample after inducing 4h, and left the heart 5 minutes, the results bacterial precipitation in 4 ℃, 5000.In precipitation, add the long-pending 1 * sds gel sample-loading buffer of 1/10 bacteria liquid, boiled ice bath 2 minutes 5 minutes.
5.SDS-PAGE Analysis and Identification is got the sample 15ul/ hole of handling, and carries out SDS-PAGE electrophoresis (spacer gel concentration is 5%, and separation gel is 12%).Behind the electrophoresis, through coomassie brilliant blue staining, methyl alcohol-glacial acetic acid destainer decolouring.By the thin layer gray scale scanning, determine the protein expression amount again.Obtain the specifically expressing band of molecular weight 32KD,, prove the Gal-9 differential protein through western blot analysis.The protein expression amount is 40%.
6. (test kit adopts GST.Bind to reclaim Gal-9 albumen through the affinity chromatography column purification
TMKitsNovagen, Meliate of Inc.an A rck KgaA, Darmstadt, Germany), and in body, the proteic antibiotic and immunization of inside and outside mensuration reorganization chicken Gal-9, discover reorganization chicken Gal-9 albumen: 1) can press down bacterium such as killing intestinal bacteria, Salmonellas, streptococcus aureus, 2) anti-avian infectious bronchitis virus, 3) after chicken is oral after 1 week, improve anti-avian infectious bronchitis virus antibody horizontal 10-20% in the immune chicken serum, improve chicken splenic lymphocyte transformation efficiency 40-50%.
Sequence table
Dash area is a target gene sequences, and all the other are carrier, is primer in the square frame, and italic is respectively initiator codon and terminator codon:
Claims (1)
1, a kind of preparation method of the chicken-beta alexin protein Gal-9 of recombinating-9 is characterized in that:
(1) according to the chicken of having delivered-beta-defensin Gal-9 gene order design Auele Specific Primer,
Upstream primer: 5 '-GGA TCC CCG GAA TTC ATG CAG ATC CTG CCT CTC-3 ',
Downstream primer: 5`-TCA GGA ATA CCA TCG GCT CCG GCA GCA GAA-3`;
(2) adopt from chicken tongue tissue, increase chicken beta-defensin Gal-9 gene and being cloned on the PMD-T carrier of RT-PCR method, get following gene order through sequencing:
TCTAAGGATCCCCGGGTACGGAGCTCGAATTCGTAATCATGGT
CATAGCTGTTTCCTGTGTGAAATTGTTATCCCGCTCACAATTCC
ACACAACATACC
Wherein: dash area is a target gene, is primer in the square frame, and italic is respectively initiator codon and terminator codon;
(3) according to chicken-beta-defensin Gal-9 gene order characteristics, with EcoRI and SalI double enzyme site with Gal-9 gene subclone in prokaryotic expression carrier PGEX-6p-1, make up recombinant expression vector PGEX-6p-1-Gal-9, with its transformed into escherichia coli BL21 competent cell, Amp+ screening positive clone;
(4) contain the single bacterium colony of e. coli bl21 37 ℃ of shaking culture in the Amp+LB substratum of positive recombinant plasmid, treat to add when the OD value reaches 0.3-0.5 IPTG, final concentration is that 0.6mM induces, induce and adopt 1ml bacterium sample after 4 hours, and in 4 ℃, 5000 left the heart 5 minutes, the results bacterial precipitation, in precipitation, add the long-pending 1 * sds gel sample-loading buffer of 1/10 bacteria liquid, boiled 5 minutes, ice bath 2 minutes carries out the SDS-PAGE Analysis and Identification, gets the sample 15ul/ hole of handling, carry out the SDS-PAGE electrophoresis, spacer gel concentration is 5%, and separation gel is 12%, behind the electrophoresis, through coomassie brilliant blue staining, methyl alcohol-glacial acetic acid destainer decolouring by the thin layer gray scale scanning, is determined the protein expression amount again, obtain the specifically expressing band of molecular weight 32KD, through western blot analysis, prove the Gal-9 differential protein, the protein expression amount is 40%;
(5) reclaim Gal-9 albumen through the affinity chromatography column purification, and in vivo, outer measure the proteic antibiotic and immunization of reorganization chicken Gal-9, discover reorganization chicken Gal-9 albumen: 1) can press down and kill intestinal bacteria, Salmonellas, streptococcus aureus, 2) anti-avian infectious bronchitis virus, 3) after chicken is oral after 1 week, improve anti-avian infectious bronchitis virus antibody horizontal 10-20% in the immune chicken serum, improve chicken splenic lymphocyte transformation efficiency 40-50%.
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| CN104388390B (en) * | 2014-11-17 | 2017-07-07 | 武汉市畜牧兽医科学研究所 | A kind of cell line of stability and high efficiency expression chicken alexin 10 and compound formulation and application |
| CN107022549B (en) * | 2017-04-19 | 2021-06-01 | 华中农业大学 | Pelteobagrus fulvidraco beta defensin gene, beta defensin antibacterial peptide and application thereof |
| CN107287203B (en) * | 2017-05-24 | 2019-10-01 | 华中农业大学 | The animal protein gene OCX-36 of optimization and its building of prokaryotic expression carrier |
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Non-Patent Citations (6)
| Title |
|---|
| Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Lynn DJ., Higgs R., Gaines S., Tierney J., James T., Lloyd AT., Fares MA., Mulcahy G., Farrelly CO.Immunogenetics,No.56. 2004 |
| Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken. Lynn DJ., Higgs R., Gaines S., Tierney J., James T., Lloyd AT., Fares MA., Mulcahy G., Farrelly CO.Immunogenetics,No.56. 2004 * |
| 广西黄鸡β-防御素Gal-4基因的克隆和体内诱导表达. 谢青梅，陈燕珊，马静云，陈俊伟，毕英佐，曹永长.中国兽医科学，第36卷第8期. 2006 |
| 广西黄鸡β-防御素Gal-4基因的克隆和体内诱导表达. 谢青梅，陈燕珊，马静云，陈俊伟，毕英佐，曹永长.中国兽医科学，第36卷第8期. 2006 * |
| 重组Gallinacin-3的克隆及在大肠杆菌中的表达. 赵建增，乔彦良，李朝阳.中国兽药杂志，第40卷第2期. 2006 |
| 重组Gallinacin-3的克隆及在大肠杆菌中的表达. 赵建增，乔彦良，李朝阳.中国兽药杂志，第40卷第2期. 2006 * |
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