CN103642816B - The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof - Google Patents
The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof Download PDFInfo
- Publication number
- CN103642816B CN103642816B CN201310655247.2A CN201310655247A CN103642816B CN 103642816 B CN103642816 B CN 103642816B CN 201310655247 A CN201310655247 A CN 201310655247A CN 103642816 B CN103642816 B CN 103642816B
- Authority
- CN
- China
- Prior art keywords
- cfsrcr
- gene
- chlamys farreri
- pcr
- albumen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to genetically engineered field, the chlamys farreri CfSRCR-2 gene of a kind of RT-PCR expression specifically and Synthesis and applications thereof.Does is the recombinant protein of CfSRCR-2 gene sequence table SEQ? ID? in NO.1 shown in aminoacid sequence.Concrete structure adopts the cDNA total length by round pcr amplification chlamys farreri CfSRCR-2 molecular gene, and carries out RT-PCR expression to its coding region.Chlamys farreri CfSRCR-2 gene provided by the invention contains the bioactive molecule of SRCR structural domain.This albumen not only can in conjunction with acetylizad low-density lipoprotein and seminose; it is a kind of important immunity receptor molecule; and directly can suppress the growth of Gram-negative bacteria; thus playing the effect of immune effector molecule, visible albumen of the present invention can be used as good immunostimulant candidate molecules.
Description
Technical field
The invention belongs to genetically engineered field, the chlamys farreri CfSRCR-2 gene of a kind of RT-PCR expression specifically and Synthesis and applications thereof.
Background technology
Immunostimulant refers to and can promote or bring out host defense, strengthens a class material of living organism resistance against diseases.Because it has the plurality of advantages such as safety, wide spectrum, efficient, nontoxic, pollution-free, low cost, at present in livestock-raising as fodder additives widespread use.But the development and application of the immunostimulant needed for the culture fishery of fast development is still in the starting stage, wherein comparatively crucial is exactly the difficult problem of the leading-in technique and methods for using them of solution active substance.
Shellfish is China's advantage sea farming object, and current annual production is close to 3,000 ten thousand tons, and account for 80% of national sea farming ultimate production, wherein bivalve shellfish output accounts for more than 95% of marine shellfish cultured output.Shellfish culture occupies very important status in China coast socio-economic development, and has huge development potentiality, but breeding lacks and the puzzlement of disease problem is the main restricting factor that industry further develops always.Start with from the immune defense system of shellfish self, illustrate gene structure and the function of important acceptor molecule, further investigated shellfish immune defence mechanism will be contributed to, for disease control and fine-variety breeding provide new approaches.
SRCR structural domain (ScavengerreceptorCys-rich) is found in scavenger receptor (Scavengerreceptor) the earliest, is one of its main functional structure, also finds this structural domain afterwards in other molecules.The molecule with SRCR structural domain is all the important acceptor in inherent immunity reaction, by identifying allogenic material and carrying out signal transmission, plays an important role in immunne response path.
Summary of the invention
The chlamys farreri CfSRCR-2 gene that the object of the invention is to provide a kind of RT-PCR to express and Synthesis and applications thereof.
For achieving the above object, the technical solution used in the present invention is:
The chlamys farreri CfSRCR-2 gene that RT-PCR is expressed, the recombinant protein of CfSRCR-2 gene is in sequence table SEQ IDNO.1 shown in aminoacid sequence.
The preparation method of the chlamys farreri CfSRCR-2 gene that RT-PCR is expressed,
1) with chlamys farreri CfSRCR-2 coding region for template, adopt P1 and P2 be that primer carries out pcr amplification, stand-by;
2) pcr amplification product is connected by T4 ligase enzyme with pEASY-E1 carrier, then will connect carry out in product conversion to e. coli bl21 (DE3) recombinant expressed;
3) the positive recombinant bacterial strain of above-mentioned structure is added IPTG and carry out inducing culture, induction obtains product, namely obtains the recombinant protein c fSRCR-2 with aminoacid sequence in sequence table SEQ IDNO.1; It is described because P1 and P2 is respectively P1:5 '-ATGCGTACACAGGACAACTTCATAT-3 '; P2:5 '-TGGTATCTCAGCCAGAACTGGACTT-3 '.
The application of the chlamys farreri CfSRCR-2 gene that RT-PCR is expressed, in described sequence table SEQ IDNO.1, the recombinant protein c fSRCR-2 of aminoacid sequence can be used as the inhibitor that preparation suppresses gram negative bacterium growth.
The advantage that the present invention has: chlamys farreri CfSRCR-2 gene provided by the invention contains the bioactive molecule of SRCR structural domain.This albumen not only can in conjunction with acetylizad low-density lipoprotein and seminose; it is a kind of important immunity receptor molecule; and directly can suppress the growth of Gram-negative bacteria; thus playing the effect of immune effector molecule, visible albumen of the present invention can be used as good immunostimulant candidate molecules.
Accompanying drawing explanation
Fig. 1 is the expression figure of the CfSRCR-2 that SDS-PAGE detects, wherein, 1, the recombination bacillus coli containing CfSRCR-2 of abduction delivering; 2, negative control; M, albumen Marker.
The chlamys farreri CfSRCR-2 that Fig. 2 provides for the embodiment of the present invention is to the restraining effect figure of fungi, gram-positive microorganism and Gram-negative bacteria growing.
Embodiment
In experimental example below, the invention will be further elaborated, but the present invention is not limited thereto.Experimental example 1
Chlamys farreri CfSRCR-2 RT-PCR express amino acid sequence is as shown in sequence table SEQ IDNO.1.
SEQIDNO.1
MRTQDNFILLVDTGLGGRIFRMDIDTQSFSPIPLNTLYTPSAFDYDPTEARIYFVDPTLKQLLSVHFSGTDIRELQQLDTNADLEKVAVDPINRVLFYADTGNDFIASVNLDGSNFKTLANDTIDEPRDLAIDPKNRVVYWTDWGASPKIEKMNYDGTGRQTIASTNMKWPNGLALDYTTNILYFVDASTDQIEKINTDGTGRSVVMSDPGSHMYGISLYKRYIYYTDWTRTSVMRVNKDGTGKTTVGPPSFKELADIHVQHYGTGMPGIVTPAPIVQDETHMFVRLVGRGNHNEGLVEVYANGVWGTICDDGWTNKDAGVICDMMGFGKANAVAVNESRYGSDGNILIFSDEVNCTGEESHIAQCTVPSDWGVHDCSHTEDAGVTCQLDPTTIRSFVLMTDSYTSEVYRMDLETGSYSAIPNANVYSPIAVDYDPVSHNVFYTDVRMGVIRQTSLDGATQKDLLQLNRISTPDGLVVDDTNRLIFYTDTGNDVIGKVGIDGTGPANIITTNLDQPRAIAVDKKNQVVYWTDWGKDPKIEKANYDGSGRQVIANGTGLNLPNGLTFDEGDQKLYFWDAGTNKIEVMNTDGSPRKVLFEDSGAHFFGLDTDEQFIYFTDWTKDGVTKIEKSGVSEIPIGPPSFVRVNGVAVYKSSSG Sequence characteristics:
◆ length: 656 amino acid
◆ type: albumen
◆ chain: strand
◆ topological framework: linear
◆ characteristic: molecular weight is 74.48kDa, iso-electric point is 4.73, and its encoding sequence has a conservative SRCR structural domain and ten low density lipoprotein receptor YWTD structural domains.
◆ source: chlamys farreri (Chlamysfarreri)
The structure of chlamys farreri CfSRCR-2 RT-PCR expression vector:
1. the structure of recombinant vectors: adopt pEASY-E1 prokaryotic expression carrier in the present invention.By round pcr, use gene-specific primer P1 (5 '-ATGCGTACACAGGACAACTTCATAT-3 ') and P2(5 '-TGGTATCTCAGCCAGAACTGGACTT-3 ') a kind of coding domain segment whole containing the molecule CfSRCR-2 gene of SRCR structural domain of amplification chlamys farreri.Reaction conditions is: first 94 DEG C of denaturations 5 minutes, then enter following circulation: 94 DEG C of sex change 30 seconds, and 60 DEG C of annealing 30 seconds, 72 DEG C extend 2 minutes, carry out 30 circulations altogether, last 72 DEG C of extensions 10 minutes.PCR primer purifying is reclaimed, is connected with pEASY-E1 carrier.After transformation of E. coli DH5 α, picking mono-clonal utilizes above-mentioned vector primer carry out bacterium colony PCR screening and check order, and after checking order-checking is correct, positive strain is carried out conservation, completes the structure of carrier.
2. the expression of recombinant protein: with BL21 (DE3) for recombinant strains, the recombinant vectors of above-mentioned structure and empty carrier are transformed into expressive host bacterium, picking mono-clonal, extract plasmid, sequence verification reading frame is correct.Picking mono-clonal, is inoculated in 50mLLB liquid nutrient medium, cultivates 12-16 hour in 37 DEG C of concussion shaking tables, is then inoculated in 200mLLB liquid nutrient medium with the volume ratio of 1:100, and 37 DEG C are cultured to OD
600=0.5-0.7.Add IPTG, make final concentration reach 1mmol/mL, 18 DEG C are continued to cultivate 12h.4 DEG C, 5000rpm, centrifugal 10min, collect thalline, frozen for subsequent use in-20 DEG C.Get 1ml bacterium liquid centrifugal, after supernatant discarded, add 80 μ L water and 20 μ L5 × albumen sample-loading buffer (250nmTris-HCl, pH6.8,10% (w/v) SDS, 0.5% (w/v) BPB, 50% (v/v) glycerine, 5% (v/v) beta-mercaptoethanol), 100 DEG C of boiling water boil 10 minutes, centrifugal, SDS-PAGE detects expression product (see Fig. 1).
3. the purifying of recombinant protein: adopted by above-mentioned gained albumen nickel sepharose FF purifying to obtain recombinant protein.
As follows with concrete operation step:
The recombinant protein of nickel sepharose FF chromatographic separation band His label:
(1) nickel sepharose FF fills post, 1.6 × 20cm, and column volume is 10ml;
(2) damping fluid 1(50mMPBS is used, pH7.4,0.5MNaCl) balance 2-5 bed volume, flow velocity is 2mLmin
-1;
(3) by 20ml cytoclasis liquid (50mMPBS, pH7.4,0.5MNaCl) 0.45 μm of membrane filtration, loading, flow velocity is 1mLmin
-1;
(4) wash 2-5 bed volume again with damping fluid 1, flow velocity is 2mLmin
-1;
(5) with respectively containing 10,20,50,100,200,300, the damping fluid 3(50mMPBS of the imidazoles of 400mM, pH7.4,0.5MNaCl) carry out stepwise elution, flow velocity is 2mLmin
-1, collect each stepwise elution peak, with the expression of SDS-PAGE detection fusion albumen;
(6) wash 5 column volumes with pure water stream, then wash 3 column volumes by the ethanol stream of 20%, flow velocity is 2mLmin
-1, pillar is placed in 4 DEG C of environment and preserves.
(7) recombinant protein of purifying needs to remove imidazoles by dialysis in suitable renaturation buffer, prevents it on the impact of protein function.Purified product at 4 DEG C of dialysis 24h, namely obtains chlamys farreri CfSRCR-2 gene recombinant protein through 50mMPBS, 100mMNaCl.
Embodiment 2
The CfSRCR-2 of restructuring suppresses the activation analysis of microorganism growth
Cell suppression test to pichia spp (Pichiapastoris), streptococcus aureus (Staphyloccousaureus), Vibrio splindidus (Vibriosplendidus): choose a strain respectively and represent bacterium (pichia spp, streptococcus aureus, Vibrio splindidus) from fungi, gram-positive microorganism and Gram-negative bacteria, wherein, pichia spp is inoculated in YPD liquid nutrient medium, S. aureus Inoculate is in LB liquid nutrient medium, and Vibrio splindidus is inoculated in 2216E substratum; Pichia spp and Vibrio splindidus 28 DEG C cultivation, streptococcus aureus 37 DEG C is cultured to OD value=0.5-0.6, by Tris-HCl damping fluid dilution OD value to 0.001, carries out bacteriostatic activity analysis.
The bacterium liquid got after 50 μ L dilutions is added in 96 porocyte culture plates, then the recombinant protein of 100 μ L is added, blank group adds 50 μ LTris-HCl(pH7.4), control group adds the BSA albumen of 50 μ L, hatch 3 hours for 18 DEG C, then add the corresponding liquid nutrient medium of 50 μ L, hatch 24 hours, 5 repetitions established by each sample.Experiment shows, blank group and control group are to pichia spp, and the growth of streptococcus aureus and Vibrio splindidus does not have restraining effect; CfSRCR-2 albumen has significant restraining effect to Vibrio splindidus, and does not have restraining effect (see Fig. 2) to the growth of gram-positive streptococcus aureus and pichia spp.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (2)
1. a chlamys farreri CfSRCR-2 gene, is characterized in that: the albumen of chlamys farreri CfSRCR-2 genes encoding is in sequence table SEQ IDNO.1 shown in aminoacid sequence.
2. chlamys farreri CfSRCR-2 gene according to claim 1 suppresses the application in the inhibitor of Vibrio splindidus growth in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310655247.2A CN103642816B (en) | 2013-12-05 | 2013-12-05 | The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310655247.2A CN103642816B (en) | 2013-12-05 | 2013-12-05 | The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103642816A CN103642816A (en) | 2014-03-19 |
| CN103642816B true CN103642816B (en) | 2015-12-02 |
Family
ID=50248102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310655247.2A Expired - Fee Related CN103642816B (en) | 2013-12-05 | 2013-12-05 | The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103642816B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619102A (en) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity |
-
2013
- 2013-12-05 CN CN201310655247.2A patent/CN103642816B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101619102A (en) * | 2008-12-16 | 2010-01-06 | 中国科学院海洋研究所 | Preparation and application of chlamys farreri peptidoglycan recognition protein (Cf-PGRP) with sterilizing activity |
Non-Patent Citations (3)
| Title |
|---|
| A novel scavenger receptor-cysteine-rich(SRCR) domain containing scavenger receptor identified from mollusk mediated PAMP recognition and binding.;Liu L et al.;《Developmental and comparative immunology》;20101029;第35卷(第2期);第227-239页 * |
| ENA Accession Number.DT716961;Wang L et al.;《ENA》;20100519;第1页 * |
| 栉孔扇贝新型清道夫受体的克隆及免疫功能研究;刘琳等;《中国动物学会、中国海洋湖沼学会贝类学会分会第十四次学会研讨会论文摘要汇编》;20091231;第101页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103642816A (en) | 2014-03-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ruiz-Lozano et al. | Identification of a putative P-transporter operon in the genome of a Burkholderia strain living inside the arbuscular mycorrhizal fungus Gigaspora margarita | |
| CN102586286B (en) | Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a | |
| Guan et al. | Brassica juncea chitinase BjCHI1 inhibits growth of fungal phytopathogens and agglutinates Gram-negative bacteria | |
| CN103848912B (en) | The recombinant protein of bay scallop peptidoglycan recognition protein and preparation thereof and application | |
| CN101906165A (en) | Expression product in series of two fish antibacterial peptide genes and expression method thereof | |
| CN103642816B (en) | The chlamys farreri CfSRCR-2 gene that a kind of RT-PCR is expressed and Synthesis and applications thereof | |
| CN107936106B (en) | Oyster containing DM9 structural domain protein CgDM9CP-4, preparation method and application | |
| CN110755605A (en) | Flavobacterium columnare transgenic engineering oral vaccine, use method and application | |
| CN104402987A (en) | Crassostrea gigas interleukin IL17N recombination protein as well as preparation and application thereof | |
| CN101565703B (en) | Eriocheir sinensis Crustin-2 gene and application of recombinant protein thereof | |
| CN105420174A (en) | Establishment of genetically engineered bacterium expressing recombined VEGF fusion protein | |
| CN103215274A (en) | Epinephelus coioides interferon IFNgamma2 and preparation method and application thereof | |
| Choi et al. | Production of an insecticidal crystal protein from Bacillus thuringiensis by the methylotroph Methylobacterium extorquens | |
| CN103319590B (en) | Application of Chlamys farreri peptidoglycan recognition protein (CfPGRP-S1) | |
| CN113185593A (en) | DM9 domain-containing recombinant protein rCgDM9CP-6 of crassostrea gigas, preparation method and application | |
| Zhou et al. | The magnetic receptor of Monascus ruber M7: Gene clone and its heterologous expression in Escherichia coli | |
| CN101955523B (en) | Preparation and application of Eriocheir sinensis whey acidic protein (WAP) with bacteriostatic activity | |
| CN102392003B (en) | Application of EDTA (ethylene diamine tetraacetic acid) in improving exocytosis volume and expression volume of escherichia coli recombinant protein | |
| CN101413007B (en) | Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses thereof | |
| CN106148381B (en) | A method of utilizing bacillus subtilis high efficient expression porcine defensin | |
| CN110117606A (en) | A kind of recombinant vector and expression of Potato Aphid effect protein Me10 gene | |
| CN105315356A (en) | Hucho taimen antimicrobial peptide HEPCIDIN as well as preparation method and application thereof | |
| CN102559672A (en) | Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof | |
| CN102372772B (en) | Ruditapes philippin RpTNF gene recombinant protein, its preparation and application | |
| Wang et al. | Analysis and expression of Pmlyzi3 from Penaeus monodon |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151202 Termination date: 20211205 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |