CN1289672C - Hippocampus protease inhibitor gene and its use - Google Patents
Hippocampus protease inhibitor gene and its use Download PDFInfo
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Abstract
The present invention relates to hippocampus protease inhibitor genes Hpi, coded albumen thereof and an application for preparing medicaments for antibiosis and antiphlogosis by the albumen. The present invention uses an RT-PCR method so as to obtain hippocampus protease inhibitor genes Hpi by separating from total RNA of hippocampi, a DNA sequence is shown as a <400>1 sequence in a sequence table, and an amino acid sequence of the gene coded albumen (recombination hippocampus protease inhibitor hkHPI) is shown as a <400>2 sequence in the sequence table. The recombination hippocampus protease inhibitor of the present invention performs functions of resisting bacteria and regulating a cell growth period, and the present invention can be used for preparing external application medicaments for treating inflammation caused by bacterial infection.
Description
Technical field
The present invention relates to a kind of Hippocampus protease inhibitor gene Hpi and encoded protein thereof, and the application of this albumen in the preparation antibacterial-anti-inflammatory drug.
Background technology
(whey acidic protein WAP) separates from the milk of rodent at first whey acid protein, because albumen is acidic protein, so gain the name.Afterwards, successively from the didelphid of camel, rabbit, pig and Marsupialia, having found the proteic existence of WAP in the milk of kangaroo, is a kind of whey-protein of extensive existence.All whey acid proteins all contain the abundant district of a plurality of halfcystines, and there are 8 halfcystines in each zone, forms four pairs of disulfide linkage centers (four-disulfide core, structural domain 4-DSC).In this structural domain, the relative position of 6 halfcystines and distance are fixed, and the relative distance of having only two halfcystines is variable.The 4-DSC structural domain is except the conservative proline(Pro) (P) in halfcystine and some positions, L-glutamic acid (G), day eastern propylhomoserin (D) and Methionin (K), and other part conservative propertys are very low.The sequential analysis of protein of all known 4-DSC of containing shows from database, and the composition of 4-DSC structural domain can be in order to following modal representation: C
1-(Xn)-C
2-(Xn)-C
3-(X5)-C
4-(X5)-C
5C
6-(X3-X5)-C
7-(X3-X4)-C
8, X represents any amino acid, n represents any amount.Near first halfcystine, also have a conserved sequence that is called as the WAP motif, KXGXCXP usually.Albumen with such constitutional features is called as the whey acid protein proteinoid, or the whey acid protein domain protein.In the member of all whey acid albumin structural domain family, a big class is the whey acid protein (WAP) that derives from mammary gland, and other one big class is the small molecules secretory protein in non-mammary gland source, mostly is proteinase inhibitor.
Along with the very fast process of gene order-checking and the submission of a large amount of est sequences, the non-mammary gland secretion albumen that contains the 4-DSC structural domain in the public database is more and more known.These albumen are expressed in each tissue, and most only have other a functional domain of a 4-DSC structural domain and some, and function is also very various, and great majority have protease inhibitory activity.At present, in human genome, 4-DSC structural domain coded message appears on some different karyomit(e)s, the albumen that wherein a lot of codings have the proteinase inhibitor function, as, prostate gland stromatin 20 (prostatestromal protein 20 on karyomit(e) 16q24, ps20), be positioned at Kallmann syndromes (the low gonad-stimulating hormone anorchia) gene on the X chromosome, be positioned at the gene cluster that contains a plurality of 4-DSC structural domain coded messages on the karyomit(e) 20q, coding elafin, secretion leukocyte protease inhibitor (SLPI), epididymis albumen (HE4) and two still unidentified protein sequences.Ps20 has only a 4-DSC structural domain, and growth inhibitory activity is arranged, and epithelial increment is had regulating effect, is considered to a potential tumor suppressor gene.Epididymis albumen HE4 is found in the epididymis of people, dog and rabbit at first, be considered to the proteinase inhibitor function at first, relevant with the decapacitation of sperm, along with going deep into of research, it is found that this gene all has expression in tissues such as respiratory tract, sialisterium and kidney, in ovarian cancer cell even occurred expressing, be the tagged molecule of ovarian cancer.In the genome of mouse, be cloned into an albuminoid of epididymis and kidney, expressing, have anti-microbial activity according to the study.That study the most deeply is SLPI and elafin, and the both is the proteinase inhibitor with anti-microbial activity, and by separating the saliva till now, its biological function is disclosed SLPI widely from first.SLPI can detect the expression of SLPI, segmental bronchus, the parotid gland, intestine and small intestine, skin, mammary gland, pancreas, female/arrenotoky road and kidney or the like by secretion or glandular epithelium cell expressing in many tissues.SLPI also has the value-added regulating and controlling effect of cell except that having with the relevant anti-inflammatory of its protease inhibitory activity, the anti-microbial activity.Zhang etc. have studied the growth regulating effect of SLPI to people's endometrial epithelial cell, find that SLPI can increase the expression of cyclin D1, and the LOX albumen of inhibition proto-oncogene p21ras and the expression of IGFBP-3, TGF-β 1 are played a part negative regulation, the high expression level of hint SLPI in epithelial cancer cells is to cause one of unusual reason of cancer cells increment.
In the big total RNA of hippocampus, separate the brand-new proteic gene of finding a coding WAP structural domain family; this albumen has 3 familial 4-DSC structural domains of WAP structural domain and WAP motif; from blast result; this albumen has the highest homology (57%) with the ovulation associated protein of separating that expression amount raises in the onset of ovulation from the salmon coelomic fluid; this ovulation associated protein also belongs to WAP domain protein family; have protease inhibitory activity and anti-microbial activity; in the salmon coelomic fluid, ovum is played a protective role, infer that this new gene is a new proteinase inhibitor.New clue and thinking is provided can for the function and the relation between the structural domain of WAP structural domain family to this proteic function research, and the potential antibacterial-anti-inflammatory drug that is developed as.
Summary of the invention
The object of the present invention is to provide a kind of new Hippocampus protease inhibitor gene and encoded protein thereof.
Another object of the present invention is to provide the application of said gene encoded protein in the preparation antibacterial-anti-inflammatory drug.
The present invention separates the Hippocampus protease inhibitor gene Hpi that obtains with the method for RT-PCR from the total RNA of male hippocampus, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.
The albumen of the invention described above genes encoding (reorganization hkHPI, called after hkHPI), its aminoacid sequence as in the sequence table＜400〉2 sequences shown in; This albumen iso-electric point is 6.55, and molecular weight is 15.4 kilodaltons.
This albumen hkHPI by expression vector pETTRX-Hpi in intestinal bacteria with soluble formal representation in the born of the same parents, expression amount can reach 140mg/L.
Described expression vector pETTRX-Hpi is by obtaining on the Kpn I/Not I site that the Hpi gene is inserted into prokaryotic fusion expression vector pETTRX (Chinese patent application number 00124832.4), it is to be to merge the companion body with the Trx, and the centre is inserted with the affinity labelling site of 6 * His and the efficient expression vector of proteolytic enzyme EK recognition site.
The selected hippocampus of the present invention belongs to sea horse (Hippocampus kuda Bleeker), picks up from hundred million and reaches continent hippocampus plant.
The structure in big hippocampus cDNA library: at first homogenate hippocampal tissue, extract total RNA.Reverse transcription is a synthetic double chain cDNA behind the chain cDNA, and double-stranded cDNA is connected back Transformed E .coli and preserves each clone's with pcDNA3.0 (available from Invitrogen company) carrier.Measure by above reorganization library clone being carried out extensive stochastic sequence, therefrom obtained the clone of 1 new coding hkHPI, called after Hpi (its dna sequence dna as in the sequence table＜400〉1 sequences shown in).New 143 amino acid whose mature peptide hkHPI of genes encoding (its aminoacid sequence as in the sequence table＜400〉2 sequences shown in), iso-electric point is 6.55, molecular weight is 15,400 dalton, feature with typical whey acid protein primary structure, intramolecularly contain 23 halfcystines, form 11 pairs of disulfide linkage and (comprise 34 pairs disulfide linkage division center territories, except that first was imperfect, two of C end were complete).
The present invention is by having designed a pair of primer, the new gene Hpi of coding hkHPI is come out from the amplification of pcDNA3.0 carrier with PCR method, be cloned on the prokaryotic fusion expression vector pETTRX, be built into expression plasmid pETTRX-Hpi, and with its transformed into escherichia coli BL21 (DE3).This expression vector (pETTRX-Hpi) is promotor with T7, adopt molecular chaperones TRX for merging the companion body, help recombinant protein correctly folding, with soluble formal representation, the C end of TRX has gentle sequence and 6 * His structure, is convenient to utilize immobilization metal part affinity chromatography to carry out purifying.Designed upstream primer contains proteolytic enzyme EK site, so that the monomeric acquisition of foreign protein.By to incubation time, induction time, the groping and optimize of conditions such as temperature, hkHPI Expression of Fusion Protein amount can reach 140mg/L, and is in solvable state fully basically.
The present invention also gropes and has optimized the proteic purification condition of reorganization hkHPI, and the ultrasonic degradation liquid of expression product is through Ni
2+Chelating Sepharose affinity chromatography obtains the higher fusion rotein of purity, and fusion rotein can obtain purity at the reorganization of the maturation more than 85% hkHPI albumen through cutting of EK proteolytic enzyme and further S100 gel filtration chromatography.
The reorganization hkHPI that the present invention obtains has the biological activity that suppresses serine protease.Recombinant protein hkHPI is added in trypsinase and the elastoser, and protease activity obviously reduces after the discovery mixing effect.In addition, recombinant protein hkHPI also has the effect of bacteria growing inhibiting, and inhibition Gram-negative that can be in various degree, the growth of gram positive bacterium can be used for preparing antibacterial-anti-inflammatory drug.
By the expression plasmid pETTRX-Hpi of hkHPI hkHPI mature polypeptide coding sequence that contains of the present invention through Kpn I/Not I double digestion, can obtain the fragment of 429bp, be hkHPI hkHPI mature polypeptide coding sequence (wherein comprising proteolytic enzyme EK recognition site).
The clone method of expression plasmid of the present invention:, press CaCl with reference to Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Labroratory Press.USA) method
2Method transforms plasmid in E.Coli.DH5 α or BL21 (DE3) bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/mL), alkaline process extracts plasmid.
Description of drawings
Fig. 1 is the proteic comparative result of several proteinase inhibitor of hkHPI albumen of the present invention and whey acid protein class, and wherein " * " expression is identical; ": " expression difference is less; ". " expression obvious difference; Complete difference is represented in the space.
Fig. 2 is the total RNA electrophoresis result of big hippocampus.
Fig. 3 is a hkHPI Hpi Gene RT-PCR electrophoresis result.1:1Kb DNA marker; 2:PCR purpose band.
Fig. 4 is that the pETTRX-Hpi expression plasmid enzyme that contains gene Hpi is cut evaluation figure.1:1Kb DNA marker; 2:pPETTRX-Hpi Kpn I/Not I double digestion.
Fig. 5 is the protein induced expression of reorganization hkHPI, affinity chromatography electrophorogram.1: through IPTG inductive pETTRX-Phi bacterial protein; 2: induce the ultrasonic supernatant total protein of bacterium; 3:Ni
2+Affinity chromatography percolation protein peak; 4:C liquid elution peak total protein; 5:D liquid elution peak total protein; 6:E liquid elution peak total protein; 7:low molecular weight marker.
Fig. 6 analyzes for the SDS-PAGE of reorganization hkHPI sieve chromatography.1: the TRX-Hpi fusion rotein that the process affinity chromatography obtains; The 2:TRX-Hpi fusion rotein cuts through EK proteolytic enzyme; 3: secondary Ni affinity chromatography; 4:S-100 molecular sieve peak (the reorganization Hpi of purifying).5：low?molecular?weight?marker。
Fig. 7 is the recombinant expression plasmid pETTRX-Hpi design of graphics that contains gene Hpi.
Embodiment
Following examples will help those of ordinary skill in the art further to understand the present invention, but not limit the present invention in any form.
Embodiment one: the extraction of the big total RNA of hippocampus and the structure in cDNA library
The extraction of total RNA is synthetic with cDNA: separate rose-red green sea anemone tentacle, adopt the Trizol method to extract the total RNA of tentacle, protein is removed in extracting.Obtaining total rna concentration is 1.04 μ g/ μ l, its A
260/ A
280=1.637, detect as seen 18s and 28s two bands clearly through 1% denaturing formaldehyde gel electrophoresis, ratio＞1 (see figure 2) shows that total RNA integrity is good.Get the total RNA of 1ug with SMART III oligonucleotide (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ') and CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)
30N
-1N-3 ') carries out synthetic first chain of reverse transcription, obtain 10 μ lcDNA, the first chain product.2 μ l, the first chain product is with 5 ' PCR primer (5 '-AAGCAGTGGTATCAACGCAGAGT-3 '), CDSIII/3 ' PCR primer (5 '-ATTCTAGAGGCCGAGGCGGCCGACATGd (T)
30N
-1N-3 ') carries out the amplification of two chains.With carrying out Sfi I cutting behind two chain protease K digestion and the purifying, connect into same Sfi I enzyme and cut on the pcDNA3.0 carrier library of handling.Random sequencing obtains the expressed sequence tag (EST) of a protease inhibitor gene Hpi, finds that through software analysis this EST is a total length.
Design of primers and RT-PCR amplification:,, design two primers with oligo primer-design software assistant analysis according to two terminal nucleotide sequences of this EST.
Upstream primer (A1): 5 '-AGG ATT CTC TCA AGA TTC A-3 '
Downstream primer (A2): 5 '-TC AGA TGA GCT CAT GAC A-3 '
Per 50 μ l PCR reaction volumes add 1 μ l cDNA strand as template, pcr amplification, electrophoresis detection, the specific amplification band (see figure 3) of expection appears in discovery near 430bp, reclaim this band, connect into T carrier random sequencing, sequence is consistent with the est sequence on the corresponding carrier library.
Embodiment two: the analysis of reorganization Hippocampus protease inhibitor gene sequence
Blast homology analysis revealed, the precursor protein of 167 amino-acid residues of Hippocampus protease inhibitor gene Hpi coding is comprising the signal peptide of 24 amino-acid residues and the maturation protein of 143 amino-acid residues.The maturation protein molecular weight is 15.4kD, and iso-electric point is 6.55, does not contain and strides the film district, at 84 amino acids places a N glycosylation site is arranged; At the 27th and 45 amino acids places a protein kinase C phosphorylation site is arranged respectively; There is a casein kinase i I phosphorylation site at 52 amino acids places; In whole protein sequence, " 4 pairs of disulfide linkage " center "s (four-disulfidecore, 4-DSC) labels of structural domain of 3 whey acid protein classes have been comprised.
It can also be seen that by Blast result, hkHPI except " skeleton of 4 pairs of disulfide linkage " center "s part and WAP family member have the higher similarity, all the other amino acid starvation homologys, on this point and the feature between other members of this family match.
The maturation protein of hippocampus putative protein enzyme inhibitors has the conservative functional domain (Fig. 1) that the WAP family member has, because this structural domain is a conserved structure that comprises 4 pairs of disulfide linkage, so claim that this structural domain is " 4 pairs of disulfide linkage " center "s (4-DSC) structural domain.8 conservative halfcystine (C that contain in the 4-DSC structural domain
1～C
8) between be in key, constitute stable rigid-skeleton, the compositional model of its disulfide linkage is as follows: SS1 (C
1, C
6), SS2 (C
2, C
7), SS3 (C
3, C
5), SS4 (C
4, C
8).Distance between each halfcystine also has its conservative property, C
1-(Xn)-C
2-(Xn)-C
3-(X5)-C
4-(X5)-C
5-C
6-(X3, X5)-C
7-(X3, X4)-C
8, X represents any amino acid, and n represents amino acid number arbitrarily, and (X3 X4) represents 3～4 amino acid.At C
3With C
8Between sequence be that conservative property is high relatively, and C
2Then mutability is higher in the position.In the comparison research that the aminoacid sequence to all members of this family carries out, find, be positioned at C
8Near the position, a conservative motif CXXP is arranged, at C
1Near a conservative motif KXGXCP is arranged, C
3To C
6Between a conservative motif CXXDXDCXGXXKCC is also arranged,, in having the family member of a plurality of these structural domains, first structural domain is imperfect sometimes, has the situation of C disappearance, but last structural domain of C end all is complete.Feature on the above WAP family member secondary structure, hippocampus putative protein enzyme inhibitors all meets, and hkHPI has 3 4-DSC structural domains, except C in first structural domain
2Outside the disappearance, the 2nd, 3 structural domains all are complete.
This shows, though similarity is on the low side between the WAP family member of hkHPI and different sources, but the conservative property on the functional domain is further confirmed us, and hkHPI is newfound WAP family member, has the activity of the proteinase inhibitor that this family member has.
Embodiment three: the structure of reorganization hkHPI expression plasmid
According to the synthetic a pair of primer of two terminal sequences of Hpi gene, upstream primer contains Kpn I and Enterokinase cleavage site, and downstream primer contains Not I cleavage site.
Upstream primer (B1):
5’-GG
G?GTA?CCG?
ACG?ACG?ACG?ACA?AAC?AGG?ATT?CTC?TCA?AGA?TTC?A-3’
Kpn?I Enterokinase?site
Downstream primer is (B2) not:
5’-ATA?AGA?AT
G?CGG?CCG?CTT?ATC?AGA?TGA?GCT?CAT?GAC?A-3’
Not?I Stop?codon
With the pcDNA3.0 plasmid that contains the Hpi gene is template, and B1, B2 are the primer PCR amplification, obtains the single band of specific amplified, and the product size is about 450bp.Pcr amplification product earlier with the combination clone of Kpn I/Not I to prokaryotic fusion expression vector pPETTRX, obtain recombinant expression vector pETTRX-Hpi (its building process is as shown in Figure 7).Expression vector pETTRX-Hpi carries out double digestion through Kpn I/Not I and identifies (enzyme is cut and be the results are shown in Figure 4).Foreign gene among the expression vector pETTRX-Hpi is identified correct through order-checking.Contain Enterokinase proteolytic enzyme cutting site in the designed upstream primer, so that excision fusion partner TRX.
Embodiment four: reorganization hkHPI Expression of Fusion Protein
With pETTRX-Hpi transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor shows that through the SDS-PAGE electrophoretic analysis thalline has tangible specifically expressing product band after inducing, molecular weight conform to theoretical value 30kD with software DNA TOOL5.1 prediction (Fig. 5).
Process is to incubation time, induced concentration, groping of conditions such as temperature: abduction delivering relatively behind the thalline enlarged culturing different time, the culture condition of genetic engineering bacterium is: the order bacterium colony is in 50ml ammonia Bian resistance LB substratum, 37 ℃, the 250rpm overnight incubation, get in the ammonia Bian resistance LB substratum that overnight culture 20ml is inoculated in 2L, 37 ℃, 250rpm is cultured to OD600=0.6 (the about 2h of enlarged culturing), add 100mM IPTG and 20% glucose to final concentration and be respectively 0.1mM and 0.2%, 25 ℃, centrifugal results thalline behind the 250rpm inducing culture 10h.Through SDS-PAGE electrophoretic analysis analysis revealed, HPI Expression of Fusion Protein amount accounts for more than 20% of bacterial protein with this understanding, almost completely is in solvable state (Fig. 5).
Embodiment five: the purifying of reorganization hkHPI fusion rotein and the acquisition of maturation protein
Total thalline of results is washed with TE (pH8.0), and (50mM pH8.0) suspends, and after the supersound process, the cracking supernatant liquor is through Ni to use Tis again
2+Chelating Sepharose affinity chromatography single step purification is analyzed and the purity of thin layer scanning analysis revealed fusion rotein reaches 80% (Fig. 5) through SDS-PAGE.With 1L genetic engineering bacterium culture is material, the final fusion rotein that obtains about 120mg purifying.
For yield and the concentration that improves recombinant protein, simplify experimental procedure, shorten treatment time to recombinant protein, with Ni2+ affinity chromatography purification of recombinant proteins the time, we adopt single stage method to carry out purifying.According to the expressed foreign protein of vector plasmid pETTRX-Hpi and the stronger characteristics of binding ability of Ni2+ affinity column, we have selected the elution requirement of simplifying for use: 50mMTris, 500mMNaCl, pH7.0 (A liquid); 50mMTris, 500mMNaCl, pH8.0 (B liquid); The 100mM imidazoles, 50mMTris, 500mMNaCl, pH8.0 (C liquid); The 150mM imidazoles, 50mMTris, 500mMNaCl, pH8.0 (D liquid); The 250mM imidazoles, 50mMTris, 500mMNaCl, pH8.0 (E liquid).Reorganization hkHPI polypeptide is eluted (Fig. 5) at E liquid.Judge the separating effect The cream of the crop from SDS-PAGE result.
Utilize Folin-phenol method to measure the protein concentration of reorganization hkHPI, the elutriant of results concentration more than 0.7mg/ml, add Enterokinase and cut, detect enzyme with SDS-PAGE and cut the result, can obviously see the bands of a spectrum (Fig. 6) of the ripe hkHPI of the representative of 20kD near.With reaction solution Ni
2+Chelating Sepharose affinity chromatography column purification (condition is the same), results contain the elution peak of ripe hkHPI, and be further purified (elutriant is PBS) with Sephacryl S100 gel permeation chromatography post, just purity at the hkHPI albumen hkHPI (Fig. 6) that recombinates of the maturation 85% or more.
Embodiment six: the inhibitor activity of reorganization hkHPI fusion rotein
With the recombinant protein hkHPI solution of embodiment five gained through G-25 sepharose chromatography column exchange buffering system, by E liquid (the 50mM Tris that contains 250mM imidazoles and 500mM NaCl, pH8.0) change 50mMTris (pH8.0) solution that contains 100mM NaCl into, and with the back a kind of solution with recombinant protein hkHPI solution dilution to 575nM.Get the good recombinant protein hkHPI solution 200 μ l of dilution, mix, placed 30 minutes for 25 ℃ with the 0.02mg/ml trypsinase of equal-volume (200 μ l).Other gets a pipe and does not add the complete active control of conduct of recombinant protein, a pipe do not add pancreatin do not add yet recombinant protein as blank.
Under 25 ℃ of conditions, in above each pipe, add 1ml BAEE substrate solution, mixing, the variation of absorbance value in the METHOD FOR CONTINUOUS DETERMINATION 5 minutes under the optical wavelength of 253nm.Enzyme of pancreatin is lived unit definition for the per minute catalytic substrate reacts, and makes the photoabsorption of 253nm increase by 0.001 needed enzyme amount.The active calculation formula of pancreatin is: U=k * V/ (0.001 * 0.2), wherein, k is A
253The slope of-time curve, V is reaction cumulative volume (ml), 0.2 is the volume (ml) of surveying pancreatin solution when living.The inhibition activity that is reduced to hkHPI of slope.The result shows that recombinant protein hkHPI has obvious restraining effect to pancreatin.
Sequence table
＜110〉Zhongshan University
＜120〉a kind of Hippocampus protease inhibitor gene and application thereof
<160>2
<210>1
<211>429
<212>DNA
＜213〉big hippocampus (Hippocampus kuda Bleeker)
<220>
<221>mat?peptide
<222>(1)…(429)
<400>1
cag?gat?tct?ctc?aag?att?cac?cca?aaa?cca?ggc?gaa?tgt?gcc?cgt?cgt 48
Gln?Asp?Ser?Leu?Lys?Ile?His?Pro?Gly?Pro?Gly?Glu?Cys?Ala?Arg?Arg
1 5 10 15
gct?tta?ctt?gca?aca?tca?aaa?aag?gga?tgt?gtg?agt?gac?caa?gac?tgc 96
Ala?Leu?Leu?Ala?Thr?Ser?Lys?Lys?Gly?Cys?Val?Ser?Asp?Gln?Asp?Cys
20 25 30
cct?ggg?gac?cac?aag?tgt?tgc?gtc?ttc?gac?tgt?gaa?gat?gtc?tgc?gtc 144
Pro?Gly?Asp?His?Lys?Cys?Cys?Val?Phe?Asp?Cys?Glu?Asp?Val?Cys?Val
35 40 45
cct?cct?tct?ttc?aat?agg?cca?gga?gtg?tgt?ccg?aat?aca?aca?ggg?cag 192
Pro?Pro?Ser?Phe?Asn?Arg?Pro?Gly?Val?Cys?Pro?Asn?Thr?Thr?Gly?Gln
50 55 60
atc?ggg?ttt?tgt?gcc?ttc?ctc?tgc?ttt?gat?gac?aga?gac?tgt?tcg?gac 240
Ile?Gly?Phe?Cys?Ala?Phe?Leu?Cys?Phe?Asp?Asp?Arg?Asp?Cys?Ser?Asp
65 70 75 80
gag?agg?aaa?tgc?tgc?cgc?aac?gga?tgt?ggt?gga?tat?aat?tgc?atg?gta 288
Glu?Arg?Lys?Cys?Cys?Arg?Asn?Gly?Cys?Gly?Gly?Tyr?Asn?Cys?Met?Val
85 90 95
cct?ttc?aaa?gtg?aag?ccg?ggc?cat?tgc?cct?ccg?ccc?gaa?gta?att?acc 336
Pro?Phe?Lys?Val?Lys?Pro?Gly?His?Cys?Pro?Pro?Pro?Glu?Val?Ile?Thr
100 105 110
acg?tgt?gca?tcg?aac?tgc?ttc?cat?gac?ggt?cag?tgt?cca?ggt?gac?cag 384
Thr?Cys?Ala?Ser?Asn?Cys?Phe?His?Asp?Gly?Gln?Cys?Pro?Gly?Asp?Gln
115 120 125
aaa?tgc?tgc?cag?aca?gcc?tgt?ggt?caa?ggc?tgt?cat?gag?ctc?atc 429
Lys?Cys?Cys?Gln?Thr?Ala?Cys?Gly?Gln?Gly?Cys?His?Glu?Leu?Ile
130 135 140
<210>2
<211>143
<212>PRT
＜213〉big hippocampus (Hippocampus kuda Bleeker)
<220>
<221>mat?peptide
<222>(1)…(143)
<400>2
Gln?Asp?Ser?Leu?Lys?Ile?His?Pro?Gly?Pro?Gly?Glu?Cys?Ala?Arg?Arg
1 5 10 15
Ala?Leu?Leu?Ala?Thr?Ser?Lys?Lys?Gly?Cys?Val?Ser?Asp?Gln?Asp?Cys
20 25 30
Pro?Gly?Asp?His?Lys?Cys?Cys?Val?Phe?Asp?Cys?Glu?Asp?Val?Cys?Val
35 40 45
Pro?Pro?Ser?Phe?Asn?Arg?Pro?Gly?Val?Cys?Pro?Asn?Thr?Thr?Gly?Gln
50 55 60
Ile?Gly?Phe?Cys?Ala?Phe?Leu?Cys?Phe?Asp?Asp?Arg?Asp?Cys?Ser?Asp
65 70 75 80
Glu?Arg?Lys?Cys?Cys?Arg?Asn?Gly?Cys?Gly?Gly?Tyr?Asn?Cys?Met?Val
85 90 95
Pro?Phe?Lys?Val?Lys?Pro?Gly?His?Cys?Pro?Pro?Pro?Glu?Val?Ile?Thr
100 105 110
Thr?Cys?Ala?Ser?Asn?Cys?Phe?His?Asp?Gly?Gln?Cys?Pro?Gly?Asp?Gln
115 120 125
Lys?Cys?Cys?Gln?Thr?Ala?Cys?Gly?Gln?Gly?Cys?His?Glu?Leu?Ile
130 135 140
Claims (4)
1. from the total RNA of hippocampus, separate the Hippocampus protease inhibitor gene Hpi that obtains with the method for RT-PCR, its dna sequence dna as in the sequence table＜400〉1 sequences shown in.
2. Hippocampus protease inhibitor gene Hpi encoded protein hkHPI as claimed in claim 1, its aminoacid sequence as in the sequence table＜400〉2 sequences shown in; This albumen iso-electric point is 6.55, and molecular weight is 15.4 kilodaltons.
3. albumen hkHPI as claimed in claim 2, it is characterized in that this albumen by expression vector pETTRX-Hpi in intestinal bacteria with soluble formal representation in the born of the same parents, expression amount reaches 140mg/L.
4. albumen hkHPI as claimed in claim 3, its expression vector pETTRX-Hpi are by obtaining on the Kpn I/Not I site that the Hpi gene is inserted into prokaryotic fusion expression vector pETTRX.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410077543 CN1289672C (en) | 2004-12-24 | 2004-12-24 | Hippocampus protease inhibitor gene and its use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410077543 CN1289672C (en) | 2004-12-24 | 2004-12-24 | Hippocampus protease inhibitor gene and its use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1644692A CN1644692A (en) | 2005-07-27 |
| CN1289672C true CN1289672C (en) | 2006-12-13 |
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