CN101928709B - Gene sequence, recombinant expression and application of hyalomma asiaticum histamine binding protein HaHBP - Google Patents
Gene sequence, recombinant expression and application of hyalomma asiaticum histamine binding protein HaHBP Download PDFInfo
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Abstract
The invention discloses a gene sequence of hyalomma asiaticum histamine binding protein HaHBP and a recombinant expression method in escherichia coli. Based on EST data of the Asian hyalomma asiaticum salivary gland differential expression gene library, the coding gene is obtained by cloning by using an RACE method, the full-length sequence of the gene is 902bp, 227 amino acid residues are coded, the predicted molecular weight of the protein is 25.9kDa, and the isoelectric point is 7.96. Bioinformatic analysis indicated that it was a histamine binding protein-homologous molecule. The gene is subcloned into a pGEX-4T-1 expression vector, transformed into BL21(DE3) host bacteria, and induced by IPTG to successfully express fusion recombinant protein, and in vitro activity experiments show that the recombinant protein has the capacity of binding histamine and can be used for preparing antihistamine drugs.
Description
Technical field
The present invention relates to the biological gene technical field, be specifically related to the gene order of hyalomma asiaticum histamine binding protein HaHBP recombinant expressed with in intestinal bacteria, and the application in the preparation antihistamine drug.
Background technology
Tick is a kind of common bloodsucker ectozoa, is again the communication media of humans and animals numerous disease.Through tick-borne pathogenic agent fungi, virus, Rickettsiae, spirochete, bacterium and protozoon etc. are arranged.Therefore, control tick and tick pass sick very important, and wherein the research and development of recombinant vaccine are research directions.Hyalomma asiaticum belongs to three host's type ticks, is distributed widely in Xinjiang, Inner Mongol, Ningxia and the each department, the Central Asia of China, can bite the mankind, can colonize on one's body the domestic animals such as ox, sheep, horse, dog, rabbit, cause the host itch pain, anaemia, become thin and symptom such as paralysis.Especially it can propagate diseases such as Xinjiang hemorrhagic fever and Q heat.
In the process of sucking blood of tick; Will inevitably cause a series of immunological rejections of host and defensive raction,, suck blood for a long time in order to overcome host's rejection; Tick sialisterium produces a large amount of bioactive molecules, and they play an important role at aspects such as anticoagulation, immunomodulatorys.Histamine binding protein (HBP) is wherein a kind of important immune modulator, reduces host's tickle and itch and inflammatory reaction thereby have antfhistamine effect, helps the whole process of sucking blood.The generation of inflammation is mediated by inflammatory mediator (like histamine and complement molecule etc.).Clinically, with the pathology disease (like irritated and digestive tract ulcer etc.) that chemicals control histamine causes, its principle is blocking histamine target cell surface H1 or H2 acceptor.The anti-inflammatory response mechanism of tick is then different, and it is to achieve the goal through salivary gland secretion and the bonded protein molecular of histamine own.Be from African Rhipicephalus appendicularis sialisterium, to separate and identified three histamine binding proteins the earliest, find that they combine characteristics with the high affine space of histamine, have accomplished the analysis (Paesen in gene clone and protein-active site subsequently; Et al.1999; 2000), after this also find similar molecule (Nuttall and Labuda, 2004) at other several kinds of tick bodies; Obviously different on the histamine binding protein structure of tick with mammalian histamine receptor; Has the height binding ability again, the plateform molecules (Mans, et al.2005) that is considered to have medically direct therapeutic value and can be used as development novel anti-inflammatory disease drug.
Summary of the invention
Technical problem to be solved by this invention is to study histamine binding protein molecule in the hyalomma asiaticum body, for design novel anti histamine bio-pharmaceutical provides the basis.
(its gene nucleotide series and corresponding amino acid sequence are seen Fig. 1 for Hyalomma asiaticumHistamine Binding Protein, HaHBP) gene order to the invention provides a kind of histamine binding protein of hyalomma asiaticum.HaHBP full length gene 902bp; 3 ' end has " polyA " and tailing signal " AATAAA "; Initiator codon and terminator codon lay respectively at 142-144bp and 823-825bp place, ORFs (ORF) total length 681bp, 227 amino acid of encoding; Predicted molecular weight is about 25.9kDa (comprising signal peptide), and iso-electric point (pI) is 7.96.Amino acids coding is carried out the signal peptide analysis, find that this aminoacid sequence has signal peptide, cleavage site is positioned at 26-27 amino acid place.Bioinformatic analysis, this molecule are the homolgous molecule of histamine binding protein.
Another object of the present invention has provided the recombinant expression method of histamine binding protein HaHBP in intestinal bacteria of hyalomma asiaticum.The present invention is according to the histamine binding protein HaHBP gene biological information science analytical results of hyalomma asiaticum; With the actual coding district subclone that does not comprise signal peptide sequence in prokaryotic expression carrier pGEX-4T-1; Make up recombinant expression plasmid pGEX-4T-1/HaHBP; After being transformed into BL21 (DE3) expression bacterium, use the IPTG abduction delivering.The SDS-PAGE electrophoretic analysis shows that the reorganization gst fusion protein obtains to efficiently express, and through the IPTG abduction delivering, fusion rotein GST-HaHBP is with inclusion body form successful expression, and size is about 49kDa, and is consistent with the expection size.
Another purpose of the present invention has provided the application of histamine binding protein HaHBP in the preparation antihistamine drug of hyalomma asiaticum.
The present invention has carried out reorganization HaHBP and has combined histamine release test; Through the Histamine-ELISA method; The result shows that HaHBP has the ability that combines histamine; And contrast GST albumen does not combine the ability of histamine, and the histamine binding protein HaHBP of hyalomma asiaticum of the present invention can be used for preparing antihistamine drug.
Description of drawings
The nucleotide sequence and the corresponding amino acid sequence of Fig. 1 hyalomma asiaticum HaHBP gene, ATG is an initiator codon, and TAC is a terminator codon, and the line aminoacid sequence is the prediction signal peptide sequence, and arrow is signal peptide cut point position.
The homology analysis of the aminoacid sequence of Fig. 2 hyalomma asiaticum HaHBP and the histamine binding protein of other ticks.AAT92202 is the histamine binding protein sequence of ixodes pacificus, and AAT66812 is the histamine binding protein sequence of ixodes scapularis.* represent all consistent amino-acid residue of three molecules, ● represent the consistent amino-acid residue of two molecules.
Fig. 3 recombinate expression and the purifying of HaHBP, 1, the HaHBP behind the purifying; 2, the reorganization tropina behind the abduction delivering; 3, no inductive reorganization tropina; M is the molecular weight of albumen standard; 4, no inductive contains pGEXT-4T-1 carrier bacterium body protein; 5, contain pGEXT-4T-1 carrier bacterium body protein after inducing; 6, the GST albumen of purifying.
The recombinate experiment that combines of HaHBP and histamine of Fig. 4.The recombinant protein of different concns or contrast GST albumen encapsulate 96 orifice plates, add the histamine of marked, chromogenic assay OD value behind the wash-out.1,2,3,4,5 to represent concentration respectively be 2,1.5,1,0.5,0.1 microgram/hole reorganization HaHBP or GST albumen.represents the experiment of HaHBP protein groups, ▲ represent the GST protein groups to test.
Embodiment
Gene clone and the sequential analysis of the histamine binding protein HaHBP of embodiment 1 hyalomma asiaticum
1. material and method
1.1 laboratory animal: hyalomma asiaticum obtains from this laboratory artificial breeding.New zealand white rabbit is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
1.2 test strain, plasmid and reagent: coli strain DH5 α, BL21 (DE3) are available from vast Imtech; Plasmid pGEM-T Easy and T4DNA ligase enzyme are available from Promega company, and TRIZOL reagent, 5 ' RACE and 3 ' RACE test kit are available from Invitrogen company; DNA glue reclaims test kit available from match Parkson company; TaqDNA polysaccharase and DNA Marker are available from TaKaRa company; Primer is synthetic by match Parkson company.
1.3 the extraction of the total RNA of hyalomma asiaticum
Get and be inoculated in the female tick of half-full blood of sucking blood on the new zealand white rabbit body 4 days, after 1 * PBS cleans, fully grind in mortar with liquid nitrogen, grinding back fully adds TRIZOL reagent, fully homogenate in homogenizer, total RNA of the female tick of the extraction half-full blood of hyalomma asiaticum;
1.4 rapid amplifying cDNA 3 ' terminal (3 ' RACE) and 5 ' terminal (5 ' RACE)
According to the est sequence (Xiang Feiyu etc. of China Agriculture Academe Shanghai Veterinary Institute through a histamine binding protein of subtractive library acquisition; Scientia Agricultura Sinica; 2006,39,2347-2353); Design 3 ' RACE and 5 ' RACE primer, 3 ' RACE-GSP:5 '-CGGCTCTGCAAGAGAACGCTCC-3 '; 3 ' RACE-Nested-GSP:5 ' CAACTTTACCTGCGAGGACAGACC-3 ' is that template increases through two-wheeled with strand cDNA, amplifies 3 ' end of full-length gene, amplification condition: 94 ℃ of preparatory sex change 5min; 94 ℃ of 45s; 53 ℃ of 40s; 72 ℃ of 1min; Total elongation 10min; 35 circulations, 5 ' RACE-GSP1:5 '-GTAGACTAGTTTCATTG-3 '; 5 ' RACE-GSP2:5 '-CTCCACCATCTGGCCGAGCATTATCG-3 '; 5 ' RACE-Nested-GSP:5 '-GGTTCGGTGCACCATACAGTGTCTG-3 '; With total RNA in the step (1) is template, with GSP1 primer synthetic cDNA first chain (sscDNA) under the reversed transcriptive enzyme effect, purifying cDNA; Add d (C) tail at cDNA then; Increase through two-wheeled through GSP2 and Nested-GSP primer again, amplify 5 ' end of full-length gene, amplification condition: 94 ℃ of preparatory sex change 4min; 94 ℃ of 45s; 55 ℃ of 40s; 72 ℃ of 1min; Total elongation 7min; 35 circulations;
1.5 the clone of goal gene
Above-mentioned steps (2) PCR product is through agarose gel electrophoresis; Reclaim test kit with DNA glue and reclaim the purpose fragment; Again the purpose fragment is connected to pGEM-T Easy carrier; And transform competent cell in DH5 α, and the method for identifying with blue hickie screening and PCR filters out positive colony and shakes the bacterium order-checking, and will check order institute's calling sequence and former sequence assembly go out total length;
1.6HaHBP Cloning of Entire Gene and sequential analysis
Obtain the HaHBP full length sequence according to step (3); The design primer; Amplify the full length sequence of HBP gene; And this full-length gene is connected to pGEM-T Easy carrier, construction recombination plasmid HaHBP-pGEM-T Easy carrier utilizes BLAST, DNAMAN (V5.2), Genetyx (v4.0) biological software to analyze the analysis of information biology such as the product size of the sequence homology of HaHBP gene, ORFs, predictive coding, iso-electric point, signal peptide: F1:5 '-CCAGTGTTTATCAAGGGTAAAGATG-3 '; R1:5 '-TAGAAATTGTACCTTTCCCACTTAC-3 ' expectation amplification size is about 820bp, amplification condition: 94 ℃ of preparatory sex change 4min; 94 ℃ of 45s; 58 ℃ of 45s; 72 ℃ of 1min; Total elongation 10min; 35 circulations;
2. result
2.1HaHBP3 ' end, 5 ' end and Cloning of Entire Gene
Through 3 ' RACE and 5 ' RACE method, 3 ' end of HaHBP gene and 5 ' end have obtained the fragment of 260bp and 400bp size respectively.Be spliced into full length sequence, size is 902bp, goes out HaHBP full length sequence size according to the full length sequence design primer amplification that splices and is 816bp (Fig. 1).
2.2HaHBP the bioinformatic analysis of full-length gene
As shown in Figure 1; HaHBP full length gene 902bp, 3 ' end has " polyA " and tailing signal " AATAAA ", and initiator codon and terminator codon lay respectively at 142-144bp and 823-825bp place; ORFs (ORF) total length 681bp; 227 amino acid of encoding, predicted molecular weight is about 25.9kDa (comprising signal peptide), and iso-electric point (pI) is 7.96.Amino acids coding is carried out signal peptide analysis (http://www.cbs.dtu.dk/services/SignalP/), find that this aminoacid sequence has signal peptide, cleavage site is positioned at 26-27 amino acid place, shows that this albumen is secreted protein.Analyze through Genetyx (v4.0), this gene is intended hydrophobic 100 of amino acids coding, and hydrophilic have 72, and neutral has 50, hydrophilic and hydrophobic amino-acid residue uniform distribution.Utilize blast to analyze HaHBP and remarkable homology is arranged with the Ixodespacificus that has reported and the HBP of two kinds of hard ticks of Ixodes scapularis; Can know between the three through Alignment and all to contain cysteine residues (C) six suitable position three sequences; Simultaneously, four to five tryptophan residues (W) are arranged (Fig. 2) in close positions.
Recombinant expressed and the active checking of the histamine binding protein HaHBP gene of embodiment 2 hyalomma asiaticums
1. materials and methods
1.1 test strain, plasmid and reagent:
Coli strain DH5 α, BL21 (DE3) are available from vast Imtech, and the T4DNA ligase enzyme is available from Promega company, and plasmid pGEX-4T-1 is available from Amersham Pharmacia Biotech; DNA glue reclaims test kit available from match Parkson company; Taq archaeal dna polymerase, DNA Marker, EcoRI and Xhol restriction endonuclease are available from TaKaRa company.GSTBind
TMTest kit is available from Novagen; Primer synthesizes histamine-HRP enzyme conjugates, K-Blue substrate, stop buffer available from Neogen company by match Parkson company.
1.2HaHBP prokaryotic expression and purifying
After the ORFs (ORF) of HaHBP gene removed signal peptide; A pair of primer is designed in both sides, coding region in remainder; F2 (introducing EcoRI restriction enzyme site GAATTC): 5 '-
GAATTCGAGAAAGTAATGGCTCACAAAGAGG-3 '; R2 (introducing Xhol restriction enzyme site CTCGAG): 5 '-
CTCGAGCGATTTCGGCAATATTTTCTTCAAT-3 '; Pcr amplification HaHBP full length gene coding region (number of writing to peptide sequence); Be cloned into the pGEX-4T-1 carrier; Construction expression recombinant plasmid HaHBP-pGEX-4T-1 accurately inserts through double digestion and order-checking evaluation purpose fragment.HaHBP-pGEX-4T-1 is transformed among the expression bacterium BL21 (DE3); 37 ℃ of 180rpm shaking tables are cultivated, and when making its OD value reach the 0.6-1.0 left and right sides, induce the GST-HaHBP expressing fusion protein with IPTG (final concentration is 1mM); Continue to cultivate about 4h and collect thalline; Cleer and peaceful deposition on the ultrasonic degradation microorganism collection through the situation of the conventional proteic expression of 12%SDS-PAGE electrophoretic analysis, is utilized GSTBind at last
TMThe purification kit purifying obtain tick histamine binding protein (Hyalomma asiaticumHistamine Binding Protein, HaHBP).
1.3 the combination histamine release test of recombinant protein
The fusion rotein GST-HaHBP (GST albumen is made negative control) that obtains with purifying spends the night for 4 ℃ in the ELISA enzyme plate as antigen coated; After the washing, add histamine-HRP enzyme conjugates, 37 ℃ of reaction 2h; Behind the thorough washing; After adding K-Blue substrate colour developing 10min, add stop buffer 5min, ELIASA is measured the OD value.
2. result
2.1HaHBP prokaryotic expression
Through the IPTG abduction delivering, fusion rotein GST-HaHBP is with insoluble inclusion body form successful expression, and size is (Fig. 3) about 49kDa, and is consistent with the expection size.Recombinant protein can obtain purified recombinant albumen through urea-denatured, renaturation and affinity chromatography.
2.2 reorganization HaHBP combines histamine release test
Through the Histamine-ELISA method; The result shows that HaHBP has the ability (it is more that the blueness of colour developing is deeply felt bright bonded histamine more) that combines histamine; And contrast GST albumen does not combine the ability (Fig. 4) of histamine, and the histamine binding protein HaHBP of hyalomma asiaticum of the present invention can be used for preparing antihistamine drug.Experimental result has confirmed that further clone's HaHBP gene is the protein-bonded new gene of coding histamine.
Sequence table
< 110>China Agriculture Academe Shanghai Veterinary Institute
< 120>gene order of a kind of histamine binding protein HaHBP of tick and recombinant expressed
< 130>specification sheets, claims
<160>1
<170>PatentIn version3.3
<210>1
<211>902
<212>DNA
< 213>nucleotide sequence of histamine binding protein HaHBP
<400>1
agtgcgtaca caacgactgt gcatggaact ggaggctcta gcccccagtg tttatcaagg 60
gtaaagatga cgtctgcttc acaatttcca taaaagggcg ccgttaagta agctttccga 120
ctagacccag aagattcgac gatggggtac agctccgcgc aacacctgat taaggccgtt 180
gttatcgctg cggcagcatc atatgttttt gtgagcgccg agaaagtaat ggctcacaaa 240
gaggagactg gcgatgacaa agttctccct attgtcagtg ttttcaacac aacttcgaaa 300
ctgtggctgt actgggaaaa tgtcacaaaa gacaataagt tatcagaaga ggaacagaca 360
ctgtatggtg caccgaacct agacttgagt gagagctgca cattcattaa aatgtttaac 420
atcagcaagg ttgacttcca cttctggtgg aaaacgataa tgctcggcca gatggtggag 480
agccattttt atggagaatt tttttcggaa ggcgggaata aaccacttgg ttcgatgaac 540
gttactgatc tttcagaaac cgaacgaaag ccttttgaaa caatgaaact agtctacagg 600
caagggcatt gcagtgtgtt ttttgtcacg gctctgcaag agaacgctcc tacagtgtgc 660
caactttacc tgcgaggaca gaccgtttca aaaaaaccgg cggaaaaatg taaggaatat 720
tacgaaaaac actgtggaac gaagattgct gtctacaatg ctacctgcaa aagagaagtc 780
aatcgagcag aaaaagaatt gaagaaaata ttgccgaaat cgtagaacaa taattgtaag 840
tgggaaaggt acaatttcta aaataaagca agtgattttg gaaaaaaaaa aaaaaaaaaa 900
<210>2
<211>227
<212>PRT
< 213>aminoacid sequence of histamine binding protein HaHBP
<400>2
Met Gly Tyr Ser Ser Ala Gln His Leu Ile Lys Ala Val Val Ile Ala
1 5 10 15
Ala Ala Ala Ser Tyr Val Phe Val Ser Ala Glu Lys Val Met Ala His
20 25 30
Lys Glu Glu Thr Gly Asp Asp Lys Val Leu Pro Ile Val Ser Val Phe
35 40 45
Asn Thr Thr Ser Lys Leu Trp Leu Tyr Trp Glu Asn Val Thr Lys Asp
50 55 60
Asn Lys Leu Ser Glu Glu Glu Gln Thr Leu Tyr Gly Ala Pro Asn Leu
65 70 75 80
Asp Leu Ser Glu Ser Cys Thr Phe Ile Lys Met Phe Asn Ile Ser Lys
85 90 95
Val Asp Phe His Phe Trp Trp Lys Thr Ile Met Leu Gly Gln Met Val
100 105 110
Glu Ser His Phe Tyr Gly Glu Phe Phe Ser Glu Gly Gly Asn Lys Pro
115 120 125
Leu Gly Ser Met Asn Val Thr Asp Leu Ser Glu Thr Glu Arg Lys Pro
130 135 140
Phe Glu Thr Met Lys Leu Val Tyr Arg Gln Gly His Cys Ser Val Phe
145 150 155 160
Phe Val Thr Ala Leu Gln Glu Asn Ala Pro Thr Val Cys Gln Leu Tyr
165 170 175
Leu Arg Gly Gln Thr Val Ser Lys Lys Pro Ala Glu Lys Cys Lys Glu
180 185 190
Tyr Tyr Glu Lys His Cys Gly Thr Lys Ile Ala Val Tyr Asn Ala Thr
195 200 205
Cys Lys Arg Glu Val Asn Arg Ala Glu Lys Glu Leu Lys Lys Ile Leu
210 215 220
Pro Lys Ser
225
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| CN119390803B (en) * | 2025-01-02 | 2025-03-04 | 鄂尔多斯市中心医院（内蒙古自治区超声影像研究所） | Tick-derived histamine binding protein HqHBP and application of encoding gene thereof in antiallergic reaction |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429235A (en) * | 2000-02-10 | 2003-07-09 | 美国国有健康与人类服务部 | Full-length infectious CDNA clones of tick borne flavivirus |
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| WO2004106369A2 (en) * | 2003-06-02 | 2004-12-09 | Evolutec Limited | Complement inhibitors from ticks |
| CN1875708A (en) * | 2002-02-07 | 2006-12-13 | 可乐丽股份有限公司 | Method of controlling acarian and device for use in the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429235A (en) * | 2000-02-10 | 2003-07-09 | 美国国有健康与人类服务部 | Full-length infectious CDNA clones of tick borne flavivirus |
| CN1875708A (en) * | 2002-02-07 | 2006-12-13 | 可乐丽股份有限公司 | Method of controlling acarian and device for use in the same |
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| WO2004106369A2 (en) * | 2003-06-02 | 2004-12-09 | Evolutec Limited | Complement inhibitors from ticks |
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