CN101538582A - Preparation method of natural antimicrobial beta-alexin 2 recombinant protein of duck - Google Patents
Preparation method of natural antimicrobial beta-alexin 2 recombinant protein of duck Download PDFInfo
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- CN101538582A CN101538582A CN200910071927A CN200910071927A CN101538582A CN 101538582 A CN101538582 A CN 101538582A CN 200910071927 A CN200910071927 A CN 200910071927A CN 200910071927 A CN200910071927 A CN 200910071927A CN 101538582 A CN101538582 A CN 101538582A
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Abstract
The invention provides a preparation method of natural antimicrobial beta-alexin 2 recombinant protein of duck. The invention uses molecular biological technique, beta-alexin (AvBD) 2 gene of duck is cloned from duck body, and proper expression host is selected to express AvBD 2 recombinant protein of duck in vitro. Further research indicates that the AvBD 2 recombinant protein of duck has the effects of broad-spectrum antibiotic activity and immunological enhancement. The recombined AvBD 2 recombinant protein of duck obtained by using the method in the invention has the following beneficial effects: 1) bacteria such as Pasteurella multocida, Bacillus subtillis, colon bacillus, salmonella, Staphylococcus aureus can be inhibited and killed; 2) the recombinant protein has very high stability for temperature and acidity-basicity and is processed for 30 min at the temperature of -20 to 100 DEG or for 30 min under the condition that pH is 3 to 12 without changing bactericidal activity.
Description
(1) 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 antibacterial action.
(2) background technology
Bird is numerous zoonosiss, and as the carrier of cause of diseases such as Salmonellas, bird flu, these cause of diseases are not only one of reason that causes the livestock product pollution, and the while is the health of serious threat people, animal also.Therefore, guarantee avian health, its innate immunity function of maximum potential ground performance, to safeguarding people, animal health, production performance of raising bird and poultry product security etc. are all significant.Antibacterial peptide is also referred to as peptide antibiotics or natural antibiotics, is the micromolecule polypeptide that a class that organism produces has broad spectrum antibiotic activity.Antibacterial peptide is very extensive in distributed in nature, is present in widely in bacterium, plant, vertebra and the invertebrates, is autarcetic important effect molecule.Because antibacterial peptide is the natural component that produces in the body, has broad-spectrum antimicrobial, antiviral and immuno-potentiation, and to body toxicological harmless, noresidue, therefore, is expected to become new additive agent for feeding and serves livestock breeding industry.Duck AvBD2 recombinant protein is naturally occurring in the duck body, contains 65 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 research of the present invention and application provide theoretical basis.
(3) summary of the invention
The bright purpose of this law is to provide has broad spectrum antibiotic activity in one, and temperature and potential of hydrogen is had the preparation method of duck beta-alexin 2 recombinant proteins of very high stability.
The object of the present invention is achieved like this:
1. according to the fowl beta-alexin gene order design Auele Specific Primer of having delivered, upstream primer: 5`-TGGCTCAGCAGATCTGCA-3`, downstream primer: 5`-CGCAATGGCAATTTATTC-3`;
2. adopt from the duck pancreatic tissue, increase duck AvBD2 gene and being cloned on the pMD-T carrier of RT-PCR method, determine that through sequencing its gene order is:
atgaggatcc?tttacctgct?cttctctgtc?cttttcctgg?tgctccaggt?ttctccagga?ttgtctttgc?cccagcggga?catgtttctc
tgtaggaaag?gctcctgcca?cttcggaaga?tgtcccatcc?acctgatcag?agttggaagc?tgctttgggt?tccgctcctg
ctgcaaatcg?ccatgggatg?tataa
3. according to duck AvBD2 gene order characteristics, with EcoRI and SalI double enzyme site with duck AvBD2 gene subclone in prokaryotic expression carrier pGEX-6p-1, make up recombinant expression vector pGEX-6p-1-AvBD2, 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 in 2-7 hour, per hour adopt 1ml bacterium sample respectively, and leaving the heart 5 minutes respectively at 4 ℃, 5000, the results bacterial precipitation adds the long-pending 1 * sds gel sample-loading buffer of 1/10 bacteria liquid in precipitation, boiled ice bath 2 minutes 5 minutes.
5. reclaim duck AvBD2 recombinant protein through the affinity chromatography column purification.
Duck AvBD2 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 30kD,, prove duck AvBD2 differential protein through western blot analysis.The protein expression amount is 40%.
Reclaim anti-microbial effect and the physicochemical property of duck AvBD2 recombinant protein through the affinity chromatography column purification at external test duck AvBD2 recombinant protein, the beneficial effect of discovering duck AvBD2 recombinant protein has: 1) can press down bacterium such as killing pasteurella multocida, Bacillus subtillis, intestinal bacteria, Salmonellas, streptococcus aureus, 2) temperature and potential of hydrogen there is very high stability, handle 30min under-20～100 ℃ of processing 30min or pH 3～12 conditions, its fungicidal activity is constant.
The present invention adopts Protocols in Molecular Biology, to duck AvBD2 gene, selects for use suitable expressive host at vivoexpression duck AvBD2 recombinant protein from the duck vivo clone.Discover that further duck AvBD2 recombinant protein has broad spectrum antibiotic activity, and temperature and potential of hydrogen are had very high stability.
(4) description of drawings
Fig. 1 is for adopting the RT-PCR method duck AvBD2 genetic expression electrophorogram photo that increases, DLMarker2000 among the figure: be followed successively by from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp, 1: duck AvBD2, M:Marker from the duck pancreatic tissue;
Fig. 2 is a duck AvBD2 expression of recombinant proteins SDS-PAGE electrophorogram photo, 1-6 is that IPTG induces the pGEX-6p-1-AvBD2 fusion rotein (30kD) of expressing behind the 2-7h among the figure, 7 is pGEX empty carrier (26kD) 8 negative contrasts, and M is protein Maker, and 9 is the pGEX-6p-1-AvBD2 of purifying; Marker: be followed successively by (KDa) from top to bottom: 35.0,25.0;
Fig. 3-Fig. 7 is a duck AvBD2 recombinant protein antibiogram, and Fig. 3 is a subtilis, and Fig. 4 is a streptococcus aureus, and Fig. 5 is a pasteurella multocida, and Fig. 6 is intestinal bacteria, and Fig. 7 is Salmonella choleraesuls;
Fig. 8-Fig. 9 is the anti-streptococcus aureus figure of duck AvBD2 recombinant protein after handling 30min under differing temps and the pH condition, and Fig. 8 is a Temperature Treatment, and Fig. 9 handles for pH value.
(5) embodiment
For a more detailed description to the present invention for example below in conjunction with accompanying drawing:
1. according to the fowl beta-defensin gene order design Auele Specific Primer of having delivered, upstream primer: 5`-TGGCTCAGCAGATCTGCA-3`, downstream primer: 5`-CGCAATGGCAATTTATTC-3`;
2. adopt the RT-PCR method duck AvBD2 gene that from the duck pancreatic tissue, increases
And be cloned on the pMD-T carrier, be defined as duck AvBD2 gene through sequencing:
atgaggatcc?tttacctgct?cttctctgtc?cttttcctgg?tgctccaggt?ttctccagga?ttgtctttgc?cccagcggga?catgtttctc
tgtaggaaag?gctcctgcca?cttcggaaga?tgtcccatcc?acctgatcag?agttggaagc?tgctttgggt?tccgctcctg
ctgcaaatcg?ccatgggatg?tataa
3. construction of prokaryotic expression vector: according to duck AvBD2 gene order characteristics, with EcoRI and SalI double enzyme site with duck AvBD2 gene subclone in prokaryotic expression carrier pGEX-6p-1, make up recombinant expression vector pGEX-6p-1-AvBD2, 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. the abduction delivering of duck AvBD2 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.Induce in 2-7 hour, per hour adopt 1ml bacterium sample respectively, and left the heart 5 minutes, the results bacterial precipitation respectively at 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 30kD,, prove duck AvBD2 differential protein through western blot analysis.The protein expression amount is 38%.
6. (test kit adopts GST.Bind to reclaim duck AvBD2 recombinant protein through the affinity chromatography column purification
TMKitsNovagen, Meliate of Inc.an A rck KgaA, Darmstadt, Germany), and, discover duck AvBD2 recombinant protein: 1) can press down bacterium such as killing pasteurella multocida, Bacillus subtillis, intestinal bacteria, Salmonellas, streptococcus aureus at the antibiotic and physicochemical property of external test duck AvBD2 recombinant protein; 2) temperature and potential of hydrogen are had very high stability, handle under 30min or pH 3～12 conditions at-20～100 ℃ and handle 30min, its fungicidal activity is constant.
The gene order table:
atgaggatcc?tttacctgct?cttctctgtc?cttttcctgg?tgctccaggt?ttctccagga?ttgtctttgc?cccagcggga?catgtttctc
tgtaggaaag?gctcctgcca?cttcggaaga?tgtcccatcc?acctgatcag?agttggaagc?tgctttgggt?tccgctcctg
ctgcaaatcg?ccatgggatg?tataa
The deduced amino acid table:
MRILYLLFSVLFLVLQVSPGLSLPQRDMFLCRKGSCHFGRCPIHLIRVGSCFGFRSCCKSP
WDV
One, application patent of invention or utility model patent must be submitted specification sheets to, duplicate (each portion of original paper and copy).
Two, specification sheets should typewrite or print, and writing should be neatly clear, and black meets ctp request, and the word height is between 0.35 centimetre to 0.45 centimetre, and line-spacing is between 0.25 centimetre to 0.35 centimetre.The specification sheets homepage is continued with this page or leaf, the available onesize blank sheet of paper suitable with quality of continuous page or leaf.Paper vertically uses, and only limit is used the front, should leave blank all around: each 2.5 centimetres at left side and top, each 1.5 centimetres of right side and bottoms.
Three, the mailing application documents must not fold.
Four, specification sheets page 1 first row should be write denomination of invention exactly, this title should with the requisition in consistent, and about placed in the middle.
Should empty delegation between denomination of invention and the specification sheets text.Should comprise following five parts on the specification sheets form, and rise at each part first row first word and to write subhead exactly that empty two lattice or newline play text behind the subhead.Example:
Technical field (body matter)
Background technology (body matter)
Summary of the invention (body matter)
Description of drawings (body matter)
Embodiment (body matter)
Specification sheets is as no accompanying drawing, and the specification sheets word segment does not just comprise description of drawings and its corresponding subhead thereof.
Five, the specification sheets word segment can have chemical formula, mathematical expression and form, but illustration must not be arranged, also publicity language must not be arranged.
Six, relate to Nucleotide or amino acid whose application, should be with this sequence table unitary part of book as an illustration, the applicant should submit to and corresponding to CD of this sequence table or floppy disk in application, and this CD or floppy disk should meet the relevant regulations of Patent Office.
Seven, specification sheets should write the page number in every page of lower frame line order placed in the middle more than two pages.
The sequence table sample
＜110〉Northeast Agricultural University
＜120〉preparation method of natural antibacterial agent duck beta-alexin 2 recombinant proteins
<140>
<141>2009-04-30
<160>1
<210>1
<211>195
<212>cDNA
＜213〉duck (Anas platyrhynchos)
<400>1
atgaggatcc?tttacctgct?cttctctgtc?cttttcctgg?tgctccaggt?ttctccagga?ttgtctttgc?cccagcggga
catgtttctc?tgtaggaaag?gctcctgcca?cttcggaaga?tgtcccatcc?acctgatcag?agttggaagc?tgctttgggt
tccgctcctg?ctgcaaatcg?ccatgggatg?tataa
Claims (1)
1, a kind of preparation method of big right antiseptic-germicide duck beta-alexin 2 recombinant proteins is characterized in that:
(1) according to the fowl beta-defensin gene order design Auele Specific Primer of having delivered, upstream primer: 5`-TGGCTCAGCAGATCTGCA-3`, downstream primer: 5`-CGCAATGGCAATTTATTC-3`;
(2) adopt from the duck pancreatic tissue, increase duck AvBD2 gene and being cloned on the pMD-T carrier of RT-PCR method, get following gene order through sequencing:
atgaggatcc?tttacctgct?cttctctgtc?cttttcctgg?tgctccaggt?ttctccagga?ttgtctttgc?cccagcggga?catgtttctctgtaggaaag?gctcctgcca?cttcggaaga?tgtcccatcc?acctgatcag?agttggaagc?tgctttgggt?tccgctcctg?ctgcaaatcgccatgggatg?tataa
(3) according to duck AvBD2 gene order characteristics, with EcoRI and SalI double enzyme site with duck AvBD2 gene subclone in prokaryotic expression carrier pGEX-6p-1, make up recombinant expression vector pGEX-6p-1-AvBD2, 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 in 2-7 hour, per hour adopt 1ml bacterium sample respectively, and respectively at 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 ice bath 2 minutes 5 minutes, carry out the SDS-PAGE Analysis and Identification, get the sample 15ul/ hole of handling, carry out the SDS-PAGE electrophoresis, spacer gel concentration is 5%, separation gel is 12%, behind the electrophoresis, through coomassie brilliant blue staining, methyl alcohol-glacial acetic acid destainer decolouring, again by the thin layer gray scale scanning, determine the protein expression amount, obtain the specifically expressing band of molecular weight 30kD, through western blot analysis, prove duck AvBD2 differential protein, the protein expression amount is 38%;
(5) reclaim duck AvBD2 recombinant protein through the affinity chromatography column purification, and, discover duck AvBD2 recombinant protein: 1) can press down bacterium such as killing pasteurella multocida, Bacillus subtillis, intestinal bacteria, Salmonellas, streptococcus aureus at the anti-microbial effect and the physicochemical property of external test duck AvBD2 recombinant protein; 2) temperature and potential of hydrogen are had very high stability, handle under 30min or pH 3～12 conditions at-20～100 ℃ and handle 30min, its fungicidal activity is constant.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433342A (en) * | 2011-12-06 | 2012-05-02 | 佛山科学技术学院 | Synthesized duck beta-defensins-2 gene, recombinant plasmid containing gene as well as production methods for recombinant yeast transformant and recombinant duck beta-defensins-2 protein |
| CN102433353A (en) * | 2011-12-06 | 2012-05-02 | 佛山科学技术学院 | Eukaryotic expression plasmid of duck AvBD2 gene, and molecular adjuvant and vaccine prepared by using plasmid |
| CN103205452A (en) * | 2011-12-06 | 2013-07-17 | 佛山科学技术学院 | Production method of recombinant yeast positive transformant of synthesized recombinant duck beta-defensin-2 protein recombinant yeast positive transformant |
| CN106177956A (en) * | 2016-08-29 | 2016-12-07 | 东北农业大学 | The method improving chicken anti-NDV virus effectiveness |
| CN108570459A (en) * | 2018-04-10 | 2018-09-25 | 南京农业大学 | A kind of method of high-efficiency fermenting production recombinant bacteria laccase |
-
2009
- 2009-04-30 CN CN200910071927A patent/CN101538582A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433342A (en) * | 2011-12-06 | 2012-05-02 | 佛山科学技术学院 | Synthesized duck beta-defensins-2 gene, recombinant plasmid containing gene as well as production methods for recombinant yeast transformant and recombinant duck beta-defensins-2 protein |
| CN102433353A (en) * | 2011-12-06 | 2012-05-02 | 佛山科学技术学院 | Eukaryotic expression plasmid of duck AvBD2 gene, and molecular adjuvant and vaccine prepared by using plasmid |
| CN102433342B (en) * | 2011-12-06 | 2013-06-12 | 佛山科学技术学院 | Synthesized duck beta-defensins-2 gene, recombinant plasmid containing gene as well as production methods for recombinant yeast transformant and recombinant duck beta-defensins-2 protein |
| CN103205452A (en) * | 2011-12-06 | 2013-07-17 | 佛山科学技术学院 | Production method of recombinant yeast positive transformant of synthesized recombinant duck beta-defensin-2 protein recombinant yeast positive transformant |
| CN103205452B (en) * | 2011-12-06 | 2014-10-15 | 佛山科学技术学院 | Production method of recombinant yeast positive transformant of synthesized recombinant duck beta-defensin-2 protein recombinant yeast positive transformant |
| CN106177956A (en) * | 2016-08-29 | 2016-12-07 | 东北农业大学 | The method improving chicken anti-NDV virus effectiveness |
| CN106177956B (en) * | 2016-08-29 | 2019-11-26 | 东北农业大学 | The method for improving the anti-NDV virus effectiveness of chicken |
| CN108570459A (en) * | 2018-04-10 | 2018-09-25 | 南京农业大学 | A kind of method of high-efficiency fermenting production recombinant bacteria laccase |
| CN108570459B (en) * | 2018-04-10 | 2022-10-04 | 南京农业大学 | Method for producing recombinant bacterial laccase by high-efficiency fermentation |
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Open date: 20090923 |