CN105087610A - Clam macrophage migration inhibitory factor gene, encoded protein of clam macrophage migration inhibitory factor gene and application of clam macrophage migration inhibitory factor gene - Google Patents
Clam macrophage migration inhibitory factor gene, encoded protein of clam macrophage migration inhibitory factor gene and application of clam macrophage migration inhibitory factor gene Download PDFInfo
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- CN105087610A CN105087610A CN201510577966.6A CN201510577966A CN105087610A CN 105087610 A CN105087610 A CN 105087610A CN 201510577966 A CN201510577966 A CN 201510577966A CN 105087610 A CN105087610 A CN 105087610A
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Abstract
The invention relates to molecular biology, in particular to a clam macrophage migration inhibitory factor gene, an encoded protein of the clam macrophage migration inhibitory factor gene and an application of the clam macrophage migration inhibitory factor gene. A cDNA sequence of the clam macrophage migration inhibitory factor gene is shown as nucleotides in the SEQ ID NO.1. A full-length cDNA sequence 804bp of the clam macrophage migration inhibitory factor gene comprises a 325bp open reading frame, and 114 amino acids are coded. Expression primers are designed according to the obtained sequence; after the full-length amplification, the expression primers are linked into expression vectors pET30a through enzyme digestion; then, obtained recombinant plasmids are transformed into escherichia coli BL21(DE)3; IPTG induction and Histrap HP affinity column purification are performed; and then, recombinant clam macrophage migration inhibitory factor protein is obtained. The clam macrophage migration inhibitory factor gene protein obtained according to the invention has oxidoreductase activity and tauromerase activity, achieves an important effect in the immune defense aspect, and can be used as shellfish growth immunopotentiators, feed, food additives and the like.
Description
Technical field
The present invention relates to molecular biology, specifically a kind of clam macrophage migration inhibitory factor gene and proteins encoded thereof and application.
Background technology
Macrophage migration inhibitory factor is a kind of multi-functional biomass cells factor, is the important regulatory factor of body innate immune responses reaction.Existing research shows, macrophage migration inhibitory factor is in institute's middle wide expression in a organized way of body.Its recombinant protein can improve the resistivity of body to bacteriological infection.Macrophage migration inhibitory factor has oxidoreductase activity and tautomerase activity simultaneously.Its oxidoreductase activity can mediate the scavenger cell and monocyte performance immunologic function that activate body, eliminates the active oxygen that immune response process produces simultaneously; Its isomerase activity site cytokine activity to this albumen is most important.Macrophage migration inhibitory factor take part in the immune response of animal, and especially for lacking the mollusk of acquired immunity, its effect is particularly important.Current test proves, and macrophage migration inhibitory factor take part in the immunne response process of biomphalaria glabrata, mitten crab and haliotis diversicolor Reeve.
Along with the sustainable development of mariculture industry, the problems such as breeding environment pollutes, disease takes place frequently allow mariculture industry suffer heavy losses.Traditional antibiotic method that utilizes controls not only to damage the medication resource of the mankind, is not also suitable for open sea water culture environment simultaneously, and therefore developing new effective immunostimulant will become one of approach of solution the farming disease harms problem.The present invention comes from the macrophage migration inhibitory factor of clam own, is a kind of native protein, can as alternative Novel immune toughener.In addition, the research in mouse show recombinant expressed macrophage migration inhibitory factor albumen can enhancing body for the resistivity of bacteriological infection.
A kind of important economic cultivated shellfish of clam, is mainly distributed in the Southeast China Sea area of China.At present, from clam, also do not obtain the report of macrophage migration inhibitory factor gene.
Summary of the invention
The object of the present invention is to provide a kind of clam macrophage migration inhibitory factor gene and proteins encoded thereof and application.
For achieving the above object, the technical solution used in the present invention is:
A kind of clam macrophage migration inhibitory factor gene, the cDNA sequence of clam macrophage migration inhibitory factor gene is for shown in SEQIDNO.1 nucleotide.
The albumen coded by cDNA of described clam macrophage migration inhibitory factor gene is in SEQIDNO.2 shown in amino acid.
An application for clam macrophage migration inhibitory factor gene, the recombination expression product of the gene of clam macrophage migration inhibitory factor shown in described SEQIDNO.1 can be used as oxydo-reductase or tautomerase.
The present invention has advantage:
The present invention screens and obtains the sequence with macrophage migration inhibitory factor gene family homology from clam EST library, based on this, adopt RACE technology and in-vitro recombination expression technology, be cloned into the cDNA full length sequence of clam macrophage migration inhibitory factor gene.By round pcr, the open reading frame sequence of amplification coding clam macrophage migration inhibitory factor is also cloned in pET30a expression vector, this albumen recombinant expressed in intestinal bacteria.Recombination expression product is after HistrapHP affinity column purifying, obtain clam macrophage migration inhibitory factor recombinant protein, this recombinant protein has oxidoreductase activity and tautomerase activity after testing, immune defense aspect plays an important role, can as shellfish growth immunostimulant and feed, food additive etc.This albumen is solubility expression, has the possibility of scale operation, can make up native protein amount low so that be difficult to the limitation of satisfying the demand.
Accompanying drawing explanation
Fig. 1 is the clam macrophage migration inhibitory factor recombinant protein of embodiment of the present invention purifying, wherein M: albumen marker; 1:0h contrasts; 4h after 2:IPTG induction; 12h after 3:IPTG induction; 4: purifying gained albumen; 5:Westernblot detects albumen.
Fig. 2 is the oxidoreductase activity that the embodiment of the present invention obtains clam macrophage migration inhibitory factor recombinant protein.Wherein MmMIF is macrophage migration inhibitory factor recombinant protein, and bovine serum albumin (BSA) is reference protein.
Fig. 3 is the tautomerase activity that the embodiment of the present invention obtains clam macrophage migration inhibitory factor recombinant protein.Wherein MmMIF is macrophage migration inhibitory factor recombinant protein, and bovine serum albumin (BSA) is reference protein.
Embodiment
In the following examples, the present invention is further elaborated, but the present invention is not limited thereto.
Embodiment 1
The cDNA sequence clone of the clam macrophage migration inhibitory factor in the present invention comprises the following steps:
1) extraction of clam total serum IgE and the purifying of mRNA;
2) from clam EST library, screening obtains the homologous sequence of macrophage migration inhibitory factor gene;
3) RACE amplification obtains the complete sequence of clam macrophage migration inhibitory factor and the checking of complete sequence.
Concrete operations are as follows:
1. the extraction of clam total serum IgE: utilize the Trizol reagent of Invitrogen company to extract total serum IgE from clam larva, utilizes the OligotexmRNA Purification Kit mRNA of QIAGENE company.
2. the clone of clam macrophage migration inhibitory factor gene cDNA full length sequence: select to design 5 ' race primer (MmMIF5'GSP & MmMIF5'NGS) and 3 ' race primer (MmMIF3'GSP & MmMIF3'NGSP) with the clam est sequence of macrophage migration inhibitory factor DNA homolog from clam EST library, carries out 3 ' and 5 ' amplification of end according to the specification sheets of race test kit.The PCR primer agarose gel electrophoresis of 1% detects, purifying and the recovery that test kit (Dalian is precious biological) carries out PCR primer is reclaimed with glue, link with pMD-19-T carrier (the precious biotech firm in Dalian) again, then transformation of E. coli competent cell Top10, select positive colony vector primer M13-47 and M13-48 to check order, acquired results, through the splicing of BioEdit software, obtains clam macrophage migration inhibitory factor full length sequence and sees SEQIDNO.1.The primer sequence used is as follows:
MmMIF5'GSP:GAGCACCAAGACTCATTACCTGAACACT
MmMIF5'NGSP：CGAACAGTCACATACGATTCTGGCTTT
MmMIF3'GSP：GGCACGACTTTCTGAGATGCTTGTATT
MmMIF3'NGSP：TACTTTCTAACTGCCATTGACTACCCA
SEQIDNO.1:
gggggttaccaagaggaagacgaacgggaaaatcgatagcgtcacaaagtcactgctttttttgcaaaaATGCCAACATTTAACATTTTTGTGTCCATACCGGCAAGTGAAATCCCAGGTGATTTTATTGGTGAAGCGTCGAAATTTATTGCAAAACAGCTTGGAAAGCCAGAATCGTATGTGACTGTTCGAGTTGTTCCTGACCAGATGATGTGTCATGGTGGAAGCAGAGACCCATGTGGCAGTGTTCAGGTAATGAGTCTTGGTGCTCTAGGACCACAGAAAAACAAAGATCATTCCTGCGCCATTGGAGAATTTCTGGAGAAGAAACTGAATATCAAGAAAGATAGATTCTACATAACGTTCTTTGACATTGCCCGCACTGACTGTGGCTATGGTGGCACGACTTTCTGAgatgcttgtattggatactaacagagacgtaatggtatccatgtctctggttccaaactccattgaatatgtttctgtagtgactctgtagcataaacacaaatacgtaaggctactcaaactggatttgaatatgtgcttattgacatattagcttgaattctacataaagtttgatatttgtaagtgtgttcatagcatatcgtggtaaaagagattaaaacatttctttgtgttttactttctaactgccattgactacccagtggtctgatctgcattttttttcagttttaagtattaagtttctttcgcagagaatgtaaaatataaaaaaaaaggaataaaaaatattaaagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
(a) sequence signature:
● length: 345bp
● type: base sequence
● chain: strand
● topological framework: linear
(b) molecule type: cDNA
C () is supposed: no
(d) antisense: no
E () is originated at first: NCBI (GenBank:GR211979.1)
(f) specificity title: clam macrophage migration inhibitory factor gene
SEQIDNO.2:
MPTFNIFVSIPASEIPGDFIGEASKFIAKQLGKPESYVTVRVVPDQMMCHGGSRDPCGSVQVMSLGALGPQKNKDHSCAIGEFLEKKLNIKKDRFYITFFDIARTDCGYGGTTF*
(a) sequence signature:
● length: 114aa
● type: aminoacid sequence
● chain: strand
● topological framework: linear
(b) molecule type: recombinant protein
C () is supposed: no
(d) antisense: no
E () is originated at first: NCBI (GenBank:AKN56918.1)
(f) specificity title: clam macrophage migration inhibitory factor albumen
The macrophage migration inhibitory factor of clam shown in SEQIDNO.1 gene, it is the cDNA sequence of cloning the macrophage migration inhibitory factor obtained from clam, this sequence 804bp, wherein entire reading frame 345bp, 5 ' non-coding region 69bp, 3 ' non-coding region 390bp, has poly-lysine price signal (AATAA) and poly-lysine tail.This gene plays an important role in the reaction of clam innate immune responses, the full length cDNA sequence of this gene is as shown in SEQIDNO.1, the albumen of its coding has the aminoacid sequence shown in SEQIDNO.2, complete coding, in vain containing 114 amino acid, comprises the functional domain that macrophage migration inhibitory factor protein family is conservative.
3. the checking of clam macrophage migration inhibitory factor gene cDNA total length: designing pair of primers cMmMIFF and cMmMIFR on the macrophage migration inhibitory factor full length sequence of order-checking splicing, take cDNA as the checking that template carries out total length.Order-checking and analysis are with step 2..The primer sequence used is as follows:
cMmMIFF：ACCAAGAGGAAGACGAACGGGA
cMmMIFR：CTGGGTAGTCAATGGCAGTTAGAAAGTA
3 ' RACE & 5 ' RACE increases reaction system used and reaction conditions:
50 μ l reaction systems, comprise:
Increase PCR response procedures used: 94 DEG C of sex change 4min, 1 circulation; 94 DEG C of sex change 30s, 60 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min, 1 circulation.
PCR reaction system (50 μ l system) and the reaction conditions of total length checking are:
Increase PCR response procedures used: 94 DEG C of sex change 4min, 1 circulation; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min, 1 circulation.
The acquisition of embodiment 2. clam macrophage migration inhibitory factor recombinant protein
Concrete operations are as follows:
The cDNA sequence corresponding according to SEQIDNO.2; design is containing Auele Specific Primer EMIF-F and EMIF-R protecting base and restriction enzyme BamHI and NotI restriction enzyme site; by the gene fragment of round pcr amplification coding clam macrophage migration inhibitory factor albumen; then being cut by enzyme is cloned in pET30a expression vector; be transformed into e. coli bl21 (DE) 3; after order-checking confirms that expression cassette is correct; inoculation positive colony is in the LB substratum containing kantlex (30mg/ml), and 37 DEG C of shaking culture are to OD
600=0.5-0.7, adding IPTG to final concentration is that 1mM induces collected by centrifugation thalline after 4 hours.Thalline with ultrasonic wave 200W 30-60 minute (3 seconds working hours, 3 seconds intermittent times) of process, makes fusion target protein discharge under condition of ice bath.Through identifying that the macrophage migration inhibitory factor fusion rotein of restructuring clam is soluble proteins.Supernatant solution after cracking is carried out purifying with certain flow rate by nickel post, and use the imidazole elution wash-out of different concns respectively, final collection contains the clam macrophage migration inhibitory factor fusion rotein of His label, and carry out SDS-PAGE electrophoresis detection purification effect, result display purifying protein band is single, and meets prediction size (19.07Kda: comprise MmeMIF (12.45kDa) & pET30a empty carrier (6.62kDa)) (Fig. 1).After utilizing PBS (0.1M) to carry out dialysis desalting, utilize protein quantification test kit, the content of quantitative analysis clam macrophage migration inhibitory factor fusion rotein is 1.3 μ g/ μ l.
The primer sequence used that increases is as follows:
EMIF-F：CGGGATCCATGCCAACATTTAACATTTTTGTGTCCA
EMIF-R：ATAAGAATGCGGCCGCGAAAGTCGTGCCACCATAGCCA
Reaction conditions is: 94 DEG C of denaturations 5 minutes; Then carry out 35 circulations and comprise 94 DEG C of sex change 30 seconds, anneal 30 seconds for 57 DEG C, 72 DEG C extend 30 seconds; Last 72 DEG C extend 10 minutes.Reaction system is identical with the composition in sequence verification PCR used reaction system in embodiment 1 and content, and difference part is only that the design of primer is different.
The oxidoreductase activity of embodiment 3. clam macrophage migration inhibitory factor recombinant protein measures
Concrete operations are as follows:
Prepare the clam macrophage migration inhibitory factor recombinant protein (1.3mg/ml) that the acquired pH of being dissolved in is the PBS damping fluid of 7.2.To comprising 1mgml
-1regular Insulin (Sigma, Chaoyang, District, Beijing, China) 2.0 μ l200mMDTT (Amresco, HaidianDistrict, Beijing are added in 100MmPBS (pH7.2) liquid, China), then the MmMIF purifying protein (pH7.2) adding 50 μ l mix.By bovine serum albumin (BSA) (Tiangen, China) in contrast albumen carry out same process.By the reaction mixture of 3 kinds of albumen in microplate reader (TECANinfiniteM200PRO, Austria), wavelength is that 630nm place different time carries out the measurement of absorbancy and draws light absorption value curve (see Fig. 2) over time.
Clam macrophage migration inhibitory factor albumen has oxidoreductase activity significantly as shown in Figure 2.
The tautomerase activity of embodiment 4. clam macrophage migration inhibitory factor recombinant protein measures
Concrete operations are as follows:
By 216 μ l4mmol/L dopachrome methyl esters (L-3,4dihydroxyphenylalaninemethylester; Sigma), 144 μ l20mmol/L sodium periodates join in the 1mmol/LEDTA (pH6.2) of 3240 μ l and form isomerase reactant.The reaction mixture mixed is reacted 30min 25 DEG C of dark places.Get the ready isomerase reactant of 0.9ml, add clam macrophage migration inhibitory factor recombinant protein (1.3mg/ml) mixing of 0.1ml, with microplate reader (TECANinfiniteM200PRO, Austria) wavelength be 475nm go out to measure reaction start after the change of light absorption value of 2-60s.Calculate the fall off rate (see Fig. 3) of the rear 60s reaction mixture absorbancy of reaction.Using BSA (Tiangen, China) in contrast albumen carry out processing as experiment contrast group equally.
Show that clam macrophage migration inhibitory factor albumen has tautomerase activity significantly by Fig. 3.
Claims (3)
1. a clam macrophage migration inhibitory factor gene, is characterized in that: the cDNA sequence of clam macrophage migration inhibitory factor gene is for shown in SEQIDNO.1 nucleotide.
2. by clam macrophage migration inhibitory factor gene according to claim 1, it is characterized in that: the albumen coded by cDNA of described clam macrophage migration inhibitory factor gene is in SEQIDNO.2 shown in amino acid.
3., by an application for clam macrophage migration inhibitory factor gene described in claim 1, it is characterized in that: the recombination expression product of the gene of clam macrophage migration inhibitory factor shown in described SEQIDNO.1 can be used as oxydo-reductase or tautomerase.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1842346A (en) * | 2001-01-12 | 2006-10-04 | 塞托凯恩药物科学公司 | Regulation of the cytotoxic lymphocyte response by macrophage migration inhibitory factor |
| CN1869207A (en) * | 2006-05-24 | 2006-11-29 | 中国科学院生物物理研究所 | Rat antihuman macrephage migration inhibiting factor monoclone antibody and its application |
| CN1972713A (en) * | 2003-08-29 | 2007-05-30 | 塞托凯恩药物科学公司 | Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor |
-
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- 2015-09-11 CN CN201510577966.6A patent/CN105087610A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1842346A (en) * | 2001-01-12 | 2006-10-04 | 塞托凯恩药物科学公司 | Regulation of the cytotoxic lymphocyte response by macrophage migration inhibitory factor |
| CN1972713A (en) * | 2003-08-29 | 2007-05-30 | 塞托凯恩药物科学公司 | Method of treatment and bioassay involving macrophage migration inhibitory factor (MIF) as cardiac-derived myocardial depressant factor |
| CN1869207A (en) * | 2006-05-24 | 2006-11-29 | 中国科学院生物物理研究所 | Rat antihuman macrephage migration inhibiting factor monoclone antibody and its application |
Non-Patent Citations (2)
| Title |
|---|
| ZOU L.等: "AKN56918.1", 《GENBANK》 * |
| ZOU L.等: "KP221196.1", 《GENBANK》 * |
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