CN103642816A

CN103642816A - Chlamys farreri scavenger receptor cys-rich (CfSRCR)-2 gene for prokaryotic recombinant expression and preparation and application thereof

Info

Publication number: CN103642816A
Application number: CN201310655247.2A
Authority: CN
Inventors: 宋林生; 刘瑞; 王玲玲; 邱丽梅; 张峘
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2013-12-05
Filing date: 2013-12-05
Publication date: 2014-03-19
Anticipated expiration: 2033-12-05
Also published as: CN103642816B

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a chlamys farreri scavenger receptor cys-rich (CfSRCR)-2 gene for prokaryotic recombinant expression and preparation and application thereof. A recombinant protein of the CfSRCR-2 gene is shown by an amino acid sequence SEQ ID NO.1 in a sequence table. A concrete building method comprises the following steps: amplifying the overall length of complementary deoxyribonucleic acid (cDNA) of a chlamys farreri CfSRCR-2 molecular gene by adopting a polymerase chain reaction (PCR) technology, and carrying out prokaryotic recombinant expression on an encoding region. The chlamys farreri CfSRCR-2 gene provided by the invention contains an active molecule of an SRCR structural domain. The protein not only can be combined with acetylated low-density lipoprotein and mannose and is an important immune receptor molecule, but also can directly inhibit growth of gram-negative bacteria, so as to play roles of immune effector molecules. The protein disclosed by the invention can be used as a good immune enhancer candidate molecule.

Description

Chlamys farreri CfSRCR-2 gene and preparation and application that a kind of RT-PCR is expressed

Technical field

The invention belongs to genetically engineered field, a kind of RT-PCR is expressed specifically chlamys farreri CfSRCR-2 gene and preparation and application.

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 safety, wide spectrum, the plurality of advantages such as efficient, nontoxic, pollution-free, low-cost, at present in livestock-raising as fodder additives widespread use.But the development and application of the immunostimulant that fast-developing culture fishery is required is still in the starting stage, and wherein comparatively crucial is exactly a difficult problem that solves the leading-in technique and methods for using them of active substance.

Shellfish is China's advantage sea farming object, and annual production at present approaches 3,000 ten thousand tons, accounts for 80% of national sea farming ultimate production, and wherein bivalve shellfish output accounts for the 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.From the immune defense system of shellfish self, start with, illustrate gene structure and the function of important acceptor molecule, will contribute to further investigated shellfish immune defence mechanism, for disease control and fine-variety breeding provide new approaches.

SRCR structural domain (Scavenger receptor Cys-rich) is found in scavenger receptor (Scavenger receptor) the earliest, is one of its main functional structure, also finds afterwards this structural domain 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, in immunne response path, plays an important role.

Summary of the invention

The chlamys farreri CfSRCR-2 gene and preparation and the application that provide a kind of RT-PCR to express are provided the object of the invention.

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 ID NO.1 shown in aminoacid sequence.

The preparation method of the chlamys farreri CfSRCR-2 gene that RT-PCR is expressed,

1) take chlamys farreri CfSRCR-2 coding region is template, and adopting P1 and P2 is that primer carries out pcr amplification, stand-by;

2) pcr amplification product is connected by T4 ligase enzyme with pEASY-E1 carrier, then connection product is transformed in e. coli bl21 (DE3), carry out recombinant expressed;

3) add IPTG to carry out inducing culture the positive recombinant bacterial strain of above-mentioned structure, induction obtains product, obtains having the recombinant protein c fSRCR-2 of aminoacid sequence in sequence table SEQ ID NO.1; It is described because P1 and P2 are 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 ID NO.1, the recombinant protein c fSRCR-2 of aminoacid sequence can be used as the inhibitor that preparation suppresses gram negative bacterium growth.

The present invention has advantages of: the bioactive molecule that chlamys farreri CfSRCR-2 gene provided by the invention contains SRCR structural domain.This albumen not only can be in conjunction with acetylizad low-density lipoprotein and seminose; it is a kind of important immunity receptor molecule; and can directly suppress the growth of Gram-negative bacteria; thereby play 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 of SDS-PAGE detection, wherein, 1, the recombination bacillus coli containing CfSRCR-2 of abduction delivering; 2, negative control; M, albumen Marker.

The restraining effect figure of the chlamys farreri CfSRCR-2 that Fig. 2 provides for the embodiment of the present invention to fungi, gram-positive microorganism and Gram-negative bacteria growing.

Embodiment

In experimental example below, the invention will be further elaborated, but the invention is not restricted to this.Experimental example 1

Chlamys farreri CfSRCR-2 RT-PCR express amino acid sequence is as shown in sequence table SEQ ID NO.1.

SEQ?ID?NO.1

MRTQDNFILLVDTGLGGRIFRMDIDTQSFSPIPLNTLYTPSAFDYDPTEARIYFVDPTLKQLLSVHFSGTDIRELQQLDTNADLEKVAVDPINRVLFYADTGNDFIASVNLDGSNFKTLANDTIDEPRDLAIDPKNRVVYWTDWGASPKIEKMNYDGTGRQTIASTNMKWPNGLALDYTTNILYFVDASTDQIEKINTDGTGRSVVMSDPGSHMYGISLYKRYIYYTDWTRTSVMRVNKDGTGKTTVGPPSFKELADIHVQHYGTGMPGIVTPAPIVQDETHMFVRLVGRGNHNEGLVEVYANGVWGTICDDGWTNKDAGVICDMMGFGKANAVAVNESRYGSDGNILIFSDEVNCTGEESHIAQCTVPSDWGVHDCSHTEDAGVTCQLDPTTIRSFVLMTDSYTSEVYRMDLETGSYSAIPNANVYSPIAVDYDPVSHNVFYTDVRMGVIRQTSLDGATQKDLLQLNRISTPDGLVVDDTNRLIFYTDTGNDVIGKVGIDGTGPANIITTNLDQPRAIAVDKKNQVVYWTDWGKDPKIEKANYDGSGRQVIANGTGLNLPNGLTFDEGDQKLYFWDAGTNKIEVMNTDGSPRKVLFEDSGAHFFGLDTDEQFIYFTDWTKDGVTKIEKSGVSEIPIGPPSFVRVNGVAVYKSSSG Sequence features:

◆ length: 656 amino acid

◆ type: albumen

◆ chain: strand

◆ topological framework: linear

◆ characteristic: molecular weight is 74.48kDa, iso-electric point is 4.73, its encoding sequence has a conservative SRCR structural domain and ten low density lipoprotein receptor YWTD structural domains.

◆ source: chlamys farreri (Chlamys farreri)

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 whole coding region fragment of molecule CfSRCR-2 gene containing SRCR structural domain of amplification chlamys farreri.Reaction conditions is: first 94 ℃ of denaturations are 5 minutes, then enter following circulation: 94 ℃ of sex change 30 seconds, and 60 ℃ of annealing 30 seconds, 72 ℃ are extended 2 minutes, carry out altogether 30 circulations, and last 72 ℃ are extended 10 minutes.PCR product purification is reclaimed, be connected with pEASY-E1 carrier.Transform after bacillus coli DH 5 alpha, picking mono-clonal utilizes above-mentioned carrier primer to carry out bacterium colony PCR screening order-checking, after checking order-checking is correct, positive strain is carried out to conservation, completes the structure of carrier.

2. the expression of recombinant protein: the BL21 (DE3) of take is recombinant strains, and the recombinant vectors of above-mentioned structure and empty carrier are transformed into expressive host bacterium, picking mono-clonal, extracts plasmid, and sequence verification reading frame is correct.Picking mono-clonal, is inoculated in 50mL LB liquid nutrient medium, in 37 ℃ of concussion shaking tables, cultivates 12-16 hour, and then the volume ratio with 1:100 is inoculated in 200mL LB liquid nutrient medium, and 37 ℃ are cultured to OD ₆₀₀=0.5-0.7.Add IPTG, make final concentration reach 1mmol/mL, 18 ℃ are continued to cultivate 12h.4 ℃, 5000rpm, centrifugal 10min, collects thalline, frozen standby in-20 ℃.Get 1ml bacterium liquid centrifugal, after supernatant discarded, add 80 μ L water and 20 μ L5 * albumen sample-loading buffer (250nm Tris-HCl, pH6.8,10% (w/v) SDS, 0.5% (w/v) BPB, 50% (v/v) glycerine, 5% (v/v) beta-mercaptoethanol), 100 ℃ of boiling water boil 10 minutes, centrifugal, SDS-PAGE detects expression product (referring to Fig. 1).

3. the purifying of recombinant protein: adopt nickel sepharose FF purifying to obtain recombinant protein in above-mentioned gained albumen.

As follows with concrete operation step:

The recombinant protein of nickel sepharose FF chromatographic separation band His label:

(1) nickel sepharose FF dress post, 1.6 * 20cm, column volume is 10ml;

(2) with damping fluid 1(50mM PBS, pH7.4,0.5M NaCl) a balance 2-5 bed volume, flow velocity is 2mL min ^-1;

(3) by 20ml cytoclasis liquid (50mM PBS, pH7.4,0.5M NaCl) 0.45 μ m membrane filtration, loading, flow velocity is 1mL min ^-1;

(4) with damping fluid 1, wash 2-5 bed volume, flow velocity is 2mL min again ^-1;

(5) with respectively containing 10,20,50,100,200,300, the damping fluid 3(50mM PBS of the imidazoles of 400mM, pH7.4,0.5M NaCl) carry out stepwise elution, flow velocity is 2mL min ^-1, collect each stepwise elution peak, with SDS-PAGE, detect the expression of fusion rotein;

(6) with pure water stream, wash 5 column volumes, then wash 3 column volumes by 20% ethanol stream, flow velocity is 2mL min ^-1, pillar is placed in 4 ℃ of environment and preserves.

(7) recombinant protein of purifying need to be removed imidazoles by dialysis in suitable renaturation buffer, prevents its impact on protein function.Purified product at 4 ℃ of dialysis 24h, obtains chlamys farreri CfSRCR-2 gene recombinant protein through 50mM PBS, 100mM NaCl.

Embodiment 2

The CfSRCR-2 of restructuring suppresses the activation analysis of microorganism growth

Growth-inhibiting experiment to pichia spp (Pichia pastoris), streptococcus aureus (Staphy loccous aureus), Vibrio splindidus (Vibrio splendidus): choose respectively a strain 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, streptococcus aureus is inoculated in LB liquid nutrient medium, and Vibrio splindidus is inoculated in 2216E substratum; 28 ℃ of cultivations of pichia spp and Vibrio splindidus, 37 ℃ of streptococcus aureuses are cultured to OD value=0.5-0.6, by Tris-HCl damping fluid dilution OD value to 0.001, carry out bacteriostatic activity analysis.

The bacterium liquid of getting after 50 μ L dilutions is added in 96 porocyte culture plates, then the recombinant protein that adds 100 μ L, blank group adds 50 μ L Tris-HCl(pH7.4), control group adds the BSA albumen of 50 μ L, hatch 3 hours for 18 ℃, then add the corresponding liquid nutrient medium of 50 μ L, hatch 24 hours, each sample is established 5 repetitions.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 the growth of gram-positive streptococcus aureus and pichia spp is not had to restraining effect (referring to Fig. 2).

Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.

Claims

1. the chlamys farreri CfSRCR-2 gene that RT-PCR is expressed, is characterized in that: the recombinant protein of CfSRCR-2 gene is in sequence table SEQ ID NO.1 shown in aminoacid sequence.

2. a preparation method for the chlamys farreri CfSRCR-2 gene that RT-PCR claimed in claim 1 is expressed, is characterized in that:

3. an application for the chlamys farreri CfSRCR-2 gene that RT-PCR claimed in claim 1 is expressed, is characterized in that: in described sequence table SEQ ID NO.1, the recombinant protein c fSRCR-2 of aminoacid sequence can be used as the inhibitor that preparation suppresses gram negative bacterium growth.