CN101748128A - Schistosoma japonica titin gene and coding protein and application thereof - Google Patents

Abstract

The invention discloses a schistosoma japonica titin gene and a coding protein and an application thereof. The schistosoma japonica titin gene comprises a nucleotide sequence shown by SEQ ID No. 1; schistosoma japonica titin comprises a protein with a nucleotide sequence shown by SEQ ID No. 2 or a protein with the same function formed by replacing and deleting or inserting the protein; and the protein is provided with a SEQ ID No. 1 sequence code. The schistosoma japonica titin is applied to schistosome vaccine preparation as an immunogen, chemical and other types medicament screening as a potential medicament action target spot and anti-schistosoma antibody preparation as a specific schistosome antigen as well as serodiagnosis; and the schistosoma japonica actin gene is applied to gene therapy.

Description

Schistosoma japonica titin gene, its proteins encoded and application

Technical field

The present invention relates to a kind of connetin, particularly relate to a kind of Schistosoma japonica titin gene, its proteins encoded and application.

Background technology

Schistosomicide is caused that by bilharzial infection 76 countries are arranged in the world, and the sufferer about 1.5 hundred million.Morbific schistosomicide has 3 kinds, both Schistosoma japonicum (Schistosoma japonicum), Schistosoma mansoni (S.mansoni) and Schistosoma haematobium (S.haematobium).At China's popular is Schistosoma japonicum.The popular of schistosomicide all causes tremendous loss for people's health and Economic development, and therefore, prevention and cure of schistosomiasis, development schistosomicide reagent for clinical diagnosis and antischistosomal medicine have huge social and economic benefit.

Can there be a lot of years in Schistosoma japonicum as parasite and not be killed by host's immunologic mechanism in human body and animal body, relevant with its immune evasion ability.Its immune evasion mechanism has antigen mimicking, antigen camouflage and immunomodulatory.Wherein immunomodulatory refers to the immunity system that (mainly being to suppress) host can be regulated, be disturbed to bilharzial albumen, makes its immune attack usefulness that can not bring into normal play, thereby helps bilharzial existence.

(Excretory/Secretory protein ESP) is exactly the protein that a class can be brought into play immunoregulation effect to bilharzial drainage/secretion antigen.These albumen are from bilharzial epidermis and gut epithelium, or the drainage/secretory of specialization.Each worm phase of schistosomicide all produces this proteinoid.ESP is potential drug target spot and vaccine molecule, thereby can disturb bilharzial immune evasion mechanism enhancing host's immunity to kill function because suppress these albumen; ESP is a potential diagnosis molecule, because these albumen are free in host's the blood, can detect with existing means easily.

Summary of the invention

Technical problem to be solved by this invention provides a kind of Schistosoma japonica titin gene, its proteins encoded and application, and this albumen can become the target molecules of schistosomicide clinical diagnosis.

For solving the problems of the technologies described above, the gene of Schistosoma japonica titin of the present invention comprises: have the nucleotide sequence shown in the SEQ IDNO.1.

Nucleotide sequence coded albumen shown in the described SEQ ID NO.1 with sequence shown in the SEQ ID NO.2.

Schistosoma japonica titin of the present invention comprises:

Have the albumen of aminoacid sequence shown in the SEQ ID NO.2 or one or more amino acid whose displacements, disappearance or insertion take place and the albumen with identical function that forms by this albumen, this albumen is the albumen with SEQ ID NO.1 sequence encoding.

Utilize the proteomics means, we have analyzed the moiety of ESP of the vitro extraction of Schistosoma japonicum in the world first.We find that the connetin (Titin) of Schistosoma japonicum is one of main component of its ESP, show that this albumen may play a significant role aspect bilharzial immunomodulatory, the immune evasion; Simultaneously, by the blood of direct analysis infection animal (rabbit), find that this albumen also exists really in host's blood, this also makes it become good diagnostic flag molecule.

Schistosoma japonica titin of the present invention is as immunogen, the application aspect the preparation blood fluke vaccine; As the potential drug target, the application aspect screening chemistry and other kind medicines; As the specificity schistosome antigen, the application aspect preparation schistosomicide antibody; Application aspect serodiagnosis; The encoding gene of Schistosoma japonica titin, the application aspect gene therapy.

Embodiment

One, method:

1, proteic separation of Schistosoma japonicum excretion and identification experiment

1). the preparation and the protein extraction of Schistosoma japonicum adult excretion product:

Schistosoma japonicum adult is separated the back in the infection animal body and is washed 2 times with 1 * PBS (phosphate buffered saline buffer), and about 800 adults suspend with 1ml PBS, and low-speed centrifugal (500rpm) is collected the supernatant sucking-off after 10 minutes, repeats 10 volumes, and supernatant is merged.With the albumen in the acetone precipitation supernatant, dissolution precipitation 50mM Tris-HCl (pH 8.3), 5mM EDTA, 1mM phenylmethyl sulfonylfluoride (PMSF), 0.5%sodium dodecylsulfate (SDS) and 0.5mM DTT, 0.5%SDS, ultrasonic short molten.

2). the proteic enzymolysis process of Schistosoma japonicum excretion:

The excretion egg white mixture spends the night with the pancreatin enzymolysis with the iodacetyl ammonia treatment of 10mM DTT and 55mM, dilution back.

3). the on-line chromatograph of Schistosoma japonicum excretion proteolysis peptide section separates and mass spectrum is identified:

The peptide mixt that enzymolysis produces pumps 30 μ l with vacuum with liquor capacity.Afterwards, on the peptide mixt of each component sample to anti-phase catch post (C18,5 μ m,

300 μ m i.d * 5mm, LC Packings), desalination under the flow velocity of 20 μ l/min.This 75 μ m * 150mm C18 post (LC Packings) of catching post and an analysis usefulness is connected then, just under the flow velocity of 200nl/min, wash-out enters the QSTAR pulser i of the electric spray ion source that Protana NanoES is housed peptide mixt from this pillar.Agilent 1100 capillary liquid phase systems (Agilent Technologies) are used to provide mobile phase A (0.5% acetic acid/water) and Mobile phase B (0.5% acetate/acetonitrile), linear gradient be in 60 minutes 5%B to 50%B, 50%B is to stop 15 minutes at 90%B to 90%B then in 30 minutes.The PicoTip that connects a 10-μ m diameter by one stainless two logical (Valco Instrument) behind analytical column receives fog-spray nozzle (New Objective), adds the spray voltage of 2500-3000 volt, to obtain stable spraying.MS/MS tandem mass spectrum spectrogram comes record by collection (IDA) and the task-circulation enhancing that information relies on.Measure in the scanning at each, choose 3-6 signal the strongest, with the ion of 2 to 3 electric charges, smash with the rolling collision energy, with the information of acquisition tandem mass spectrum.

4). the proteic software search of Schistosoma japonicum excretion is identified:

The database of mass spectrum search usefulness comprises the Schistosoma japonicum protein sequence of downloading from public database NCBI from 11717 (time is on October 16th, 2008).Search software is MASCOT (http://www.matrixscience.com/, Matrix Science).Search parameter is: maximum leakage point of contact, 3; Monoisotopic; The peptide charge number ,+1/+2/+3; Modify halfcystine carbamoylmethylation and methionine oxidation.After Search Results merges, carry out filtering screening: 1. to the search result list of each tandem mass spectrum spectrogram, only extract the result of ranking, other then abandon at first with following standard; 2. if in the peptide section that search obtains KR is arranged, KK, RK, sequences such as RR, then this peptide section abandons; 3. search for the peptide section that obtains, length is 8 reservations more than the amino acid, less than 8 amino acid whose abandoning; 4. repeat the peptide section got to, only calculate that wherein score value is the highest, these peptide section score values are added up, as proteic score; 5. proteic score surpasses 60, just thinks believable result.The low albumen that divides keeps after manual the inspection or abandons.Match host or bovine trypsin, keratic peptide section, then abandon.

Carry out the search of reverse protein sequence library simultaneously.Oppositely protein pool is with the counter-rotating of Schistosoma japonicum protein sequence, keep length, form constant.According to forward storehouse and the result that reverse library searching obtains, determine false positive rate.Method of calculation: false positive=2* (oppositely storehouse peptide hop count)/(forward storehouse peptide hop count+reverse storehouse peptide hop count).False positive is controlled at 0%.

2, the excretion albumen of direct analysis Schistosoma japonicum from infection animal serum

1. schistosoma japonicum cercariae infects rabbit, collects and infects the 42nd day the animal serum in back.Form with the liquid chromatography-mass spectrography analysing protein behind the serum protein enzymolysis.The Protein Data Bank of mass-spectrometric data search Schistosoma japonicum is according to above-mentioned standard screening peptide section.The peptide section that obtains is removed the peptide section of any coupling with the genes database (comprising mRNA, EST, gene order-checking data etc.) of Blast search rabbit, and the peptide section that remains all is that schistosomicide is distinctive.Utilize the peptide section then, search obtains corresponding schistosomicide protein sequence.

2. albumen abundance quantitative analysis: (Spectra count SC), represents the abundance height with this to calculate the mass spectrum sum that each Identification of Fusion Protein obtains.

Two, result

1) the proteic proteomics qualification result of the external excretion of Schistosoma japonicum

Identify 110 schistosomicide albumen altogether.Wherein connetin is one of higher albumen of relative abundance, and the SC sum is 32, and peptide section score summation is 1736, identifies this proteic 5 kinds of peptide sections (table 1) altogether.This explanation, connetin is one of main protein ingredient in the Schistosoma japonicum adult excretion product.Adult is in host (as the people) body, and its excretion product must be discharged in host's the blood circulation system, therefore, detects the existence of schistosomicide excretion product, just can detect schistosomicide.And the high albumen of abundance in the excretion product, the easiest being detected, therefore, connetin is suitable clinical diagnosis molecule, can utilize existing universal method such as ELISA to detect schistosomicide characteristic protein in patient's blood sample easily.

The mass spectrum qualification result in external excretion protein groups of table 1 Schistosoma japonica titin

2) we directly identify the excretion albumen of Schistosoma japonicum in the world first from infected animal serum.Identify a kind of peptide section (table 2) of connetin, proved that the connetin of Schistosoma japonicum is present in the serum of host animal really, can be detected, thereby can become the target molecules of schistosomicide clinical diagnosis.

The qualification result of table 2 Schistosoma japonica titin in infection animal serum

Sequence table

＜110〉Research Center of Shanghai Human Genome

＜120〉Schistosoma japonica titin gene, its proteins encoded and application

<130>NP-08-12792

<160>2

<170>PatentIn?version?3.3

<210>1

<211>3325

<212>RNA

＜213〉Schistosoma japonicum (Schistosoma japonicum)

<400>1

cctttttagt?tgataaatta?aattaaaatt?tgaggtttat?tggtaataat?ataatgtcaa 60

ctgtagctat?gccagcaact?gtttttcaag?catctactcc?tggaaatcct?ccacaaattt 120

taagtccatt?atgtggtggc?ggtgaagttc?cagaaggaga?acctgttcat?ttagaattac 180

gaattgatcc?tccaggtgat?ttgcaggtac?aatggtttaa?agatggtgtt?gcattaagtg 240

caggttctcg?ttacaatact?atgtgtgaac?gtggtttagc?ctcattggat?gtaatatata 300

caatccagga?agacagtggt?acctattttt?gtgtgatatc?gaatccatat?ggacatgctc 360

aaagtgaagc?agttcctgtg?aatgttatac?ctgaaattga?tccaaatcaa?gatcatttag 420

atgcacaatt?tgctgatcta?tatcaagcaa?ctagtgcacc?tataaaagaa?tatgaacgtg 480

atcagagaag?tcagccaaag?agtgcaccac?aattttctca?gcaaccggtg?attagtagta 540

gtgatgttct?tgaaggtgaa?cctgtgagaa?tggaggcata?cttaaatacg?acaggtgatt 600

caacactaca?agttgattgg?atgaaaaatg?gacaaccagt?tggtacagga?agtcgtttta 660

acgccatcct?tgatagaggt?ttagttgtcc?tggaaattgg?ttattgtctg?gtcgatgata 720

gtggattgta?tgtttgtgta?gctacaaatg?cacttggacg?cacagagagc?caacctgtgg 780

aactaagatg?ccaaccagaa?gaacgaattg?ttactacctc?tatcctggat?cagcaatcaa 840

taaatcattt?gaagcaatta?gaggaaccag?gagaagattt?cgatcatagt?gccaatgcaa 900

caattcaagg?tatatcccca?tctttcaagg?ccaatttaga?acctgctacg?gttcaagtta 960

atgaggatga?acctgtaatt?ttctccgtac?cggttgactg?tggaaatgga?gatcacgtgg 1020

ttattgagtg?gaaacgtgat?ggtcaggttg?ttaaaacagg?atctcgaatc?actggaagtt 1080

tagagcttgg?aatagccagc?ttaaaaattc?attatgccca?gccgaatgat?actggagcat 1140

acacttgtca?tatttctaca?gaatatggat?cagctgactg?tggtccatcc?aacttattat 1200

gtgaagctac?tggtagtata?atcgctgcaa?gccaacttcc?aggtgacaaa?gaaaaaggtc 1260

tactagcgat?tgaagcgata?gaagcgaatt?tgcatgcacc?acgcggtaca?gtatgggaag 1320

atgaaagtcc?acatgaagca?cctgttatta?ttcaacaacc?acaatctgtt?ggtgaaattg 1380

aggaaggaac?agccattcat?tttgatgttc?aggtagaacc?cgcttctgat?ccttcactta 1440

ttgttgaatg?gtttaaaagt?ggcaatttat?taacaagtgg?aacaagattc?aaagttgcat 1500

atgatagagg?attagccact?ctagatattt?tgtatactat?tcctgaagat?agcggtgaat 1560

attggtgccg?tgtagagaat?aaagctggtc?aaatggaaag?taatcatgta?caagttttat 1620

gtaacgccag?tgcgtctgta?atcacacaca?gtaatctttt?acaaggtaca?gagggctaca 1680

atttaataaa?agctattgaa?gaagttgaac?agattgacgg?agatgaatat?agatacatag 1740

aggacgaaga?ggttgattca?gctcctcatt?ttgatgtcaa?gcctcaagca?gctactatag 1800

gtgaaggttc?cccagttaaa?tttttagtac?gtgttagtgg?taaaccaaca?ccacagctta 1860

cttggtatct?caatgacgaa?ccaattgaac?aagattcgat?tacgaaaatc?tatagtgatg 1920

gtgcaataaa?ttacttagaa?atgagtcgtt?gccatgcttt?acaaggaact?aataagctac 1980

atgttattgc?acaaaatcaa?ttaggaaaag?ctgaagctga?aacggttctt?acagttgtat 2040

tggcggaaga?tttcagaccg?gatttaaaac?atgttaaacc?agaaaatcct?tacaaaaaga 2100

tggttggatt?acgtaaagta?gactgttcac?cagaattaaa?taaagcgtta?acgagaacta 2160

aaccgtctgc?tcaaacaatt?ttagaaatgg?aaagaggttc?tgaaatgaaa?gctcgtactt 2220

atcgttcacc?agaggtagtc?gaggcagaga?aaatgttgga?tcaacttgct?tcgagtttaa 2280

gaaaatctga?acttaaacga?ccattggtaa?atggtagcca?tcaaaatgat?gcagcataac 2340

ctattcacat?agcattgaaa?tgcattacta?ttcatactta?aatcaaatat?ctacttgatt 2400

attctatttc?ccaatgttta?tttactgaac?aaacaattaa?acttttgtgc?ttccaaatat 2460

aaaaaaaaaa?gattataaac?ataaatgatg?aaatatcaaa?cattaattgt?tttattatca 2520

catgtttaca?tttatattct?ttgcttttaa?atgaaattta?tcagtttcta?tcatttaatc 2580

aatagtttct?tttaataata?cttatttcag?aacatttatt?taaatgatga?tatatgactt 2640

tgaaataaat?tttattattc?attactgaat?aatacaatgg?taatgatgaa?tgcattcatt 2700

acttgtaatc?tgatccgata?cttttcttta?ttcttagaat?tctttggttg?cttatattca 2760

gtcaattaca?tttgtttgtt?tatgtgtagt?ttttgtttct?tgatttaaaa?aaaaatgttt 2820

attacttacc?gatgaaatta?attcagtact?actattatta?tgaaatgaaa?attgaatcta 2880

tacttcatat?tataacttaa?taattaatat?ctaaatgata?attcaaacca?tttgtgaata 2940

actgaatgaa?tgaaaaatta?aattacttaa?ttttaatgac?aatatttatg?ttattacaat 3000

gttatccccc?attttatgga?tattgcatta?tttaaaatgt?tcaatttatg?ctgttatatt 3060

gtttatttaa?cagttcacct?ttaaatctac?ttttattcat?gaagaaaaaa?caaaagaaga 3120

tagaatacat?tatcatcagt?ataaaaatat?aaatcattta?ttgaatatgt?atcactggtt 3180

acaatgattc?ataccttaaa?gatcataatt?aagcattcta?aatgtgttag?ggattctttc 3240

tattatattc?tgtaacagta?aattatacac?taaggctatc?atacaaagta?tatacttgta 3300

ttattatgaa?aaaaaaaaaa?aaaaa 3325

<210>2

<211>761

<212>PRT

＜213〉Schistosoma japonicum (Schistosoma japonicum)

<400>2

MSTVAMPATV?FQASTPGNPP?QILSPLCGGG?EVPEGEPVHL?ELRIDPPGDL?QVQWFKDGVA 60

LSAGSRYNTM?CERGLASLDV?IYTIQEDSGT?YFCVISNPYG?HAQSEAVPVN?VIPEIDPNQD 120

HLDAQFADLY?QATSAPIKEY?ERDQRSQPKS?APQFSQQPVI?SSSDVLEGEP?VRMEAYLNTT 180

GDSTLQVDWM?KNGQPVGTGS?RFNAILDRGL?VVLEIGYCLV?DDSGLYVCVA?TNALGRTESQ 240

PVELRCQPEE?RIVTTSILDQ?QSINHLKQLE?EPGEDFDHSA?NATIQGISPS?FKANLEPATV 300

QVNEDEPVIF?SVPVDCGNGD?HVVIEWKRDG?QVVKTGSRIT?GSLELGIASL?KIHYAQPNDT 360

GAYTCHISTE?YGSADCGPSN?LLCEATGSII?AASQLPGDKE?KGLLAIEAIE?ANLHAPRGTV 420

WEDESPHEAP?VIIQQPQSVG?EIEEGTAIHF?DVQVEPASDP?SLIVEWFKSG?NLLTSGTRFK 480

VAYDRGLATL?DILYTIPEDS?GEYWCRVENK?AGQMESNHVQ?VLCNASASVI?THSNLLQGTE 540

GYNLIKAIEE?VEQIDGDEYR?YIEDEEVDSA?PHFDVKPQAA?TIGEGSPVKF?LVRVSGKPTP 600

QLTWYLNDEP?IEQDSITKIY?SDGAINYLEM?SRCHALQGTN?KLHVIAQNQL?GKAEAETVLT 660

VVLAEDFRPD?LKHVKPENPY?KKMVGLRKVD?CSPELNKALT?RTKPSAQTIL?EMERGSEMKA 720

RTYRSPEVVE?AEKMLDQLAS?SLRKSELKRP?LVNGSHQNDA?A 761

Claims (8)

1. the gene of a Schistosoma japonica titin is characterized in that: comprising: have the nucleotide sequence shown in the SEQ ID NO.1.

2. the gene of Schistosoma japonica titin as claimed in claim 1 is characterized in that: the albumen of sequence shown in the nucleotide sequence coded SEQ of the having ID NO.2 shown in the described SEQ IDNO.1.

3. Schistosoma japonica titin comprises: have the albumen of aminoacid sequence shown in the SEQ ID NO.2 or one or more amino acid whose displacements, disappearance or insertion take place and the albumen with identical function that forms by this albumen.

4. the application of Schistosoma japonica titin aspect the preparation blood fluke vaccine.

5. the application of Schistosoma japonica titin aspect screening of medicaments.

6. the application of Schistosoma japonica titin aspect preparation schistosomicide antibody.

7. the application of Schistosoma japonica titin aspect serodiagnosis.

8. a Schistosoma japonica titin utilizes its encoding gene, the application aspect gene therapy.