CN101475942A - B19 virus VP1 unique region gene - Google Patents

Abstract

The invention discloses a B19 virus VP1 unique region gene, and constructs a B19 procaryon to express pQE30-VP1 unique region of cloning plasmid, express and purify the recombinant protein; the recombinant protein can cause immune cells response in vitro, and produce the highly effective antibodies; the B19 virus recombination protein of the present invention can be used for antigen diagnosis and for preparing antibody and vaccine for prevention of B19 virus.

Description

A kind of B19 virus VP1 unique region gene

Technical field

The invention belongs to biological technical field, relate to the clone of B19 virus gene, particularly a kind of clone of new gene fragment of B19 virus VP1 distinct zones, and make up prokaryotic expression carrier, expression and purification obtains to have immunogenic recombinant protein VP1 distinct zones.

Background technology

1.B19 virus and pathogenic

(Human Parvovirus B19 is Cossart YE in 1975 with serendipitous in No. 19 sample during hepatitis B virus in the countercurrent immunoelectrophoresis screening healthy blood donor blood PVB19) to the human parvovirus B19, hence obtains one's name.It is present unique virus that causes human diseases that oneself knows during the Parvoviridae parvovirus belongs to, and also is the simplest dna virus of volume minimal structure in the animal virus.1981, in the children that suffer from sicklemia and aplastic anemia, detect this virus, thereby confirmed that it is pathogenic, not only in erythema infectiosum (but also claiming erythema infectiosum), thrombopenic purpura, joint disease, myocarditis, pericarditis, acute hepatitis patient, detect this virus in succession later on, but also find that this virus can pass through the placental infection fetus, cause pregnant woman's miscarriage, premature labor or fetus edema, stillborn foetus.1987 in 1 routine congenital immunodeficiency syndrome anemia people alleged occurrence have chronic B19 virus to infect.The existence that all has chronic persistence B19 virus to infect in the receptor who studies show that in AIDS patient, organ transplantation subsequently, the immunosuppressed patients such as tumour patient of chemotherapy.In recent years, confirm that also B19 virus infection and mucocutaneous lymphnode syndrome, one cross the minimizing of property protoerythrocyte, systemic lupus erythematous, dermatomyositis, chronic autoimmunity thrombocyte/neutrophilic granulocytopenia, hepatitis that some is serious, relevant by diseases such as the kidney damage of immunocomplex mediation and germinal cell tumor of testis, even can cause septicemia.

Zhang Guocheng equals nineteen ninety and detect the B19 virus specific antibody first from the women of child-bearing age and children, confirms that China exists B19 virus to infect.Take the lead in having launched the research of aspects such as relation, B19 virus diagnosis of infection, treatment and prevention subsequently at home, find successively and reported that B19 virus is one of China pregnant woman non-immunity miscarriage, children acute important cause of disease of hindering diseases such as crisis, the purple paralysis of acute thrombocytopenic again B19 virus and disease.Cardiac muscular tissue's sample of the embryonic tissue of spontaneous abortion and congenital heart infant is carried out B19 virus DNA to Zhang Guocheng etc. and IgM detects, prompting B19 virus intrauterine infection may with the certain relation of having of spontaneous abortion and congenital heart disease; And pass through PCR, RT-PCR and ISH equimolecular biology techniques detect the B19 virus gene in the congenital heart disease cardiac muscular tissue, discovery has the high positive rate, it mainly is distributed in the cardiac cell nucleus, the prompting B19 virus may be the important cause of disease of congenital heart disease morbidity, participate in expression of nucleic acids and influenced the growth of heart, caused heart malformations.

The route of transmission of B19 virus is mainly by respiratory tract, blood products and mother and baby's vertical transmission, and the viremia that height is tired increases the possibility through blood or blood product propagation.In voluntary blood donor, the recall rate of B19 virus is 1:167 to 1:35000, mainly is distributed in 17-45 year, and is the most obvious with 1-39 year.In China, the recall rate of B19 virus is 5.08% among the blood donor.

2.B19 the constitutional features and the somatotype of virus

The B19 virus particle is made up of the single strand dna of two capsid proteins and a wire, is symmetric icosahedro three-dimensional arrangement, no coating, diameter 22-24nm, average 23nm.Visible ghost and complete virion in Electronic Speculum and negative staining, sophisticated infectious virion molecular weight is 5.6 * 10 ⁶Dalton, wherein DNA accounts for half of full weight.The B19 virus gene is the little DNA of strand, total length 5596bp, and two ends have the long tumor-necrosis factor glycoproteins of 383bp to form compound hairpin structure, and G, C content height have strong homology, are the primers of synthetic whole piece chain.The compound hairpin structure at two ends makes that B19 virus gene secondary structure is firm, and is difficult for being cloned in the bacterium.Two big open reading frame are arranged in the B19 virus genome, almost extend whole genome, the open reading frame in left side (406-2501bp) coding Nonstructural Protein (NS), two the capsid protein VP1 of open reading frame coding and the VP2 on right side.The amino acid coding of VP2 (3088-5093bp) is included in the amino acid coding (2416-5093bp) of VP1, and few 226 the amino acid whose amino tails of its ratio of components latter are so these two kinds of capsid proteins have identity on amino acid is formed.Do not comprise the proteic zone of VP2 in the VP1 albumen and be called VP1 distinct zones protein region.VP1 and VP2 form capsid and are wrapped in B19 virus DNA surface, constitute the antigenicity of B19 virus, and wherein 95% is made up of VP2, and VP1 only accounts for 5%.In virus main and epitope be positioned at VP1 distinct zones and VP1-VP2 joining region.Though the amount of VP1 is lacked than VP2, the VP1 distinct zones is most important for forming sophisticated B19 virus capsomere, and most of fragment of VP1 extends to the capsid outside, is the major antigen that stimulates body generation neutrality antibody, easier and antibodies.

1986, Shade etc. obtained the genome nucleotide sequence (Au strain) of the nearly total length of B19 virus first.Subsequently, Blundell MC separates the mensuration (Wi strain) of having carried out full length sequence from asymptomatic blood donor's PVB19 virus strain to a strain.People such as Hicks in 1996 have also carried out the mensuration (Stu strain) of full length sequence from asymptomatic blood donor's PVB19 virus strain to another strain separation.Gallinella in 1999 have announced the full genome nucleotide sequence of a PVB19 virus strain (HV strain) again.Mori etc. have adopted methods analyst that restriction endonuclease map analyzes 48 strain virus according to the difference of restriction enzyme mapping, are divided into 4 types with virus.Subsequently, Umene etc. have repeatedly carried out restriction analysis to the B19 virus strain, find a new genotype: promptly exist only in the V-type among the relevant encephalopathic patient of B19 virus.The distribution of different genotype exists time and geographical difference, and national popular virus strain such as America and Europe belongs to the II type mostly, and the IV type only limits to Japan, and other country does not see popular.Kerr etc. make SSCP (single strand conformationpolymorphism, single strand conformation polymorphism) somatotype to 50 strain B19 virus and discover that the III type of SSCP banding pattern mainly is distributed in Japan and Britain.Umene etc. have studied the somatotype of 1981 and 1986-1987 Japan B19 virus epidemic strain respectively, find that variation has taken place virus strain, and 1981 is the IV type, and 1986-1987 is II (type).Erdman etc. discover 29 strain B19 virus, homology is higher to each other for the 3 strain B19 virus (XA strain) in China Xi'an, compare with 26 strains from other country, no matter still on amino acid levels, all there are significant difference, enzyme spectrum analysis method to show that this 3 strain belongs to V-type basically at nucleotide level.

3.B19 the sudden change of virus

Erdman DD etc. adopt the methods such as polymorphism analysis of order-checking, restriction endonuclease analysis or PCR product single chain polypeptide, and the gene of B19 is carried out Variability Analysis, disclosed the B19 sequence and had variation, but variability is less, only is 6%.Hemauer etc. find that the NS gene is the most conservative in the gene of B19, play an important role in the duplicating of virus, and VP1 and VP2 show bigger variability, are about 2-3%.Hicks etc. have measured the nearly full length sequence of B19 virus Wi strain and Stu strain respectively, and contrast with the Au strain sequence of Shade, have 46 bases as a result in the genome of these two kinds of virus strain variation has taken place, wherein 19 bases are in the NS1 district, 27 bases are in the VP district, the base variation of B19 is converted to the master with base, and insertion and deletion mutantion are rare.The 23 strain B19 virus VP1 distinct zones that Haseyama etc. collect in the period of to 1986-1997 are carried out nucleotide sequencing, and make comparisons with the Au strain, and the result shows that having 14 bases changes, and causes 4 amino acid whose changes; The aberration rate of Nucleotide is 0.3-2.7% in all virus strain, and amino acid whose aberration rate is 1.0-3.0%.

Zhang Guocheng etc. study the variation of Chinese strain B19 virus gene, the domestic B19 virus VP1 of 1 strain coding region partial nucleotide sequence (bp2699-2955) analyzed with restriction enzyme digestion and dna sequencing technology in 1994, find this coding region and external Au strain basically identical, only have a Nucleotide point mutation, the corresponding amino acid of coding does not change; Calendar year 2001 is used long segment PCR, technology such as overlapping PCR and nucleotide sequencing, China's B19 virus epidemic strain is analyzed, and by computer and external B19 virus type strain comparison, the result discloses from the geographic China in Xi'an B19 virus epidemic strain B19-XA2 strain and compares with foreign standard Au strain, 4 coding mutations are arranged, and cause 2 amino acid changes of coding; Adopted round pcr from the infant serum of 2 routine Chinese aplastic anemia, to amplify VP1 unique region gene fragment in 2002, compare with external Wi strain distinct zones sequence behind the sequencing analysis, there are 2 place's Nucleotide to change, and can cause coded amino acid to change; We adopted round pcr to amplify 3 B19 virus Chinese strain B19-XA17 from the infant serum of Chinese aplastic anemia in 2003, the NS gene fragment of XA18 and XA20, compare with the nucleotide sequence of external Au pnca gene behind the sequencing analysis, 4 coding mutations are arranged, and cause that 4 coded amino acid change.

Jiang Xiaoling has equaled to measure in 2003 B19 Guangdong strain isolated VP1/VP2 (2909-3806bp) the gene segment sequence that 2 strains (C1 strain, C2 strain) separate from acute lymphoblastic leukemia and hinder the patient again, compare with type strain (AU strain) corresponding sequence, the C2 strain has 32 bases variation has taken place, the nucleotide diversity rate is 3.56%, wherein 19 are the base conversion, and 13 is transversion, does not have the disappearance and the insertion of base, base changes and has caused 6 amino acid whose changes, and aberration rate is 2.01%; The C1 strain has 10 bases that change has taken place, and wherein 7 are the base conversion, and 3 is transversion, and base changes and only caused 1 amino acid whose change.

Zhao Guoqiang equals employing round pcrs in 2005 full gene of segmentation amplification B19 virus from 1 routine B19 male miscarriage patients serum, obtain 7 fragments that overlap each other, it is cloned into the T carrier respectively, carry out sequencing analysis, B19-ZZ1 total length 5594bp Nucleotide as a result, wherein 2754bp C → T and 3204bpA → G undergo mutation; The B19 VP1 unique region gene variation result who separates order-checking with Zhang Guocheng etc. from 1 routine acute spy's property sent out thrombopenia purpura infant is in full accord.

The variation of B19 virus is relevant with its persistent infection, and the variation of strain is bigger in B19 virus persistent infection person body.Umene etc. study the nucleotide sequence of 10 strain B19 virus, find that its reason is viral persistent infection from the sequence change of the sequence change of 3 strain virus of stillborn foetus tissue B19 virus strain in other patient body.Kerr etc. have carried out for up to 26-85 month follow up a case by regular visits to the patient of 53 routine B19 virus acute infections.After following up a case by regular visits to end, there are 7 routine patient B 19 viral DNAs positive, DNA to B19 virus before and after wherein 4 examples are followed up a case by regular visits to carries out the PCR-SSCP analysis revealed, there is the SSCP banding pattern of 2 examples that very big difference is arranged before and after following up a case by regular visits to, prompting is through persistent infection with after repeatedly duplicating, and bigger variation has taken place the B19 virus base.And occur adhering to separately the two strain B19 virus coexistence of different genetic pedigrees sometimes in the patient body that infects of chronic lasting B19 virus, even the phenomenon of re-constituted appears.But also have the scholar to think that genovariation and persistent infection are irrelevant, Gallinella etc. have carried out nucleotide sequence analysis to many strains B19 virus of persistent infection person 16 months and 1 month respectively, find that variation is rare.

4.B19 virogene reorganization, protein expression and vaccine research

The B19 virus separation difficulty can not be grown on traditional substratum, and it can only duplicate on normal people's medullary cell, people's megalokaryocyte, leukemia cell system cells such as (MB-02).The CFU-E of people's marrow and later stage, inmature red corpuscle was the main target cell of its invasion and attack, and virus is duplicated in nucleus and made cytolysis, hinders erythropoiesis.

Because B19 virus is different from other common human virus, can not be on traditional substratum growth and breeding, the antigen source is very difficult, therefore tends in recent years with gene recombination B19 virus structural protein as diagnostic antigen.The antigenic production of B19 virus that develops into of Protocols in Molecular Biology provides new way, utilize recombinant DNA technology to obtain B19 virus DNA, express B19 virus reorganization glutelin and Nonstructural Protein, significant for mechanism of causing a disease, development diagnostic reagent and the preventative vaccine etc. of further discussion B19 virus gene structure and variation, B19 virus.

Confirmations such as Manaresi utilize the reorganization glutelin that contains VP1 and VP2 or only contain VP2, by the ELISA method, all can detect the immune response to B19 virus structural protein conformation and natural linear epitope.Erdman etc. with the B19 virus Detection of antigen of Chinese hamster ovary cell expression 400 parts of samples, and separate antigen with serum and compare, prove that antigen expressed can replace separating antigen and carry out antibody test.The single peptide of the B19 virus structural protein VP1 distinct zones of usefulness escherichia coli expressions such as Soderlund carries out ELISA as antigen and detects, and compares with complete virus antigen, and its susceptibility is respectively 83% and 87%, and specificity is respectively 94% and 90%.VP1 and VP2 albumen can be used as the candidate molecules of vaccine, but NS1 albumen can not be as the candidate molecules of vaccine.

Li Zhihong, Zhang Guocheng equals to deliver " proteic expression of people microvirus B19VP1 distinct zones and purifying " at " cell and molecular immunology magazine " in 2007, utilize prokaryotic expression system construction recombinant plasmid VP1-PQE30, transform successfully back bulk fermentation culturing bacterium, the VP1 distinct zones albumen of expressing human microvirus B19, purity of protein reaches more than 95% behind the purifying.

Summary of the invention

The object of the present invention is to provide the new gene fragment of a kind of B19 virus VP1 distinct zones, and make up prokaryotic expression carrier, expression and purification obtains that immunogenic recombinant protein VP1 distinct zones is arranged, the VP1 of existing B19 virus contrast exists significantly and distinguishes among this fragment and the Genbank.

For achieving the above object, the present invention is by the following technical solutions:

With the blood specimen DNA that comprises B19 virus is template, right according to B19 virus gene order design VP1 distinct zones primer, adopts the amplification of PCR method; Described primer is to as follows:

Forward primer F:aaggatccat gagtaaagaa agtggcaaat g

Reverse primer R:agtcgactca cagttggcta tacctaaagt cat

This primer is to the about 480bp of target sequence of amplification, and BamH I restriction enzyme site is introduced in the upstream, and Sal I restriction enzyme site is introduced in the downstream; By target sequence and plasmid pQE30 being carried out BamHI, SalI double digestion, construction recombination plasmid pQE30-VP1 distinct zones.

The recombinant plasmid transformed competence colibacillus bacterium, choose bacterium cultivation back extraction plasmid and carry out BamHI and SalI double digestion, 1% agarose gel electrophoresis, visible clear band in the 480bp place, recombinant plasmid pQE30-VP1 distinct zones successfully constructs, and VP1 distinct zones sequence is shown in SEQ ID NO.1.

Induce Recombinant Protein Expression by IPTG, the recombinant protein of expressing by affine, ion-exchange, the multiple chromatography method purifying of hydrophobic and gel-filtration, obtain purification of recombinant proteins, adopt SDS-PAGE, Western blot to identify recombinant protein, the visible clear band in the 22kDa place, recombinant protein successfully constructs, and its aminoacid sequence is shown in SEQ ID NO.2.

Recombinant protein is carried out lymphocyte transformation experiment, immunocompetence mensuration, and experimental result shows that the recombinant protein that is obtained can stimulate mouse to produce at the proteic antibody of VP1 distinct zones in experiment in vitro, and antibody titer reaches 1:8000.

Compared with prior art, the immunization experiment of recombinant protein pQE30-VP1 distinct zones can cause at B19 virus produce immunne response and produce antibody illustrated success of the present invention the clone B19 virus VP1 unique region gene, detect through BLAST and to show that VP1 unique region gene provided by the invention is new gene.Because B19 virus is in different areas and the time exists variation and the variation of B19 virus is relevant with its pathogenic prognosis, the new gene of VP1 distinct zones provided by the invention, made up B19 prokaryotic expression pQE30-VP1 distinct zones cloned plasmids, and express, be purified into the recombinant protein that can cause immunne response, for the diagnostic antigen that new B19 virus is provided, mouse out the B19 virus variation law, the treatment of B19 virus and the preparation of vaccine produce positive meaning.

Description of drawings

Fig. 1 is a recombinant plasmid pQE30-VP1 distinct zones double digestion gel electrophoresis qualification result;

Fig. 2 is recombinant protein pQE30-VP1 distinct zones Western Blot qualification result figure;

Fig. 3 is BP1 virus VP 1 distinct zones sequence B LAST analytical results figure.

Embodiment

The present invention adopts Protocols in Molecular Biology, clone the proteic new gene of B19 virus VP1 distinct zones, made up recombinant plasmid pQE30-VP1 distinct zones and also expressed successfully, the recombinant protein pQE30-VP1 of purifying can cause the immunne response at B19 virus.The present invention adopts Li Zhihong, Zhang Guocheng to equal the feasibility of disclosed in " cell and molecular immunology magazine " " proteic expression of people microvirus B19VP1 distinct zones and purifying " process in 2007, with the DNA of blood specimen recently that comprises B19 virus is the screening source, specifically by realizing that following scheme is achieved.

The structure of 1 recombinant plasmid (pQE30-VP1 distinct zones)

1.1 right according to B19 virus gene order design VP1 distinct zones primer, adopt the amplification of PCR method; Described primer is to as follows:

Forward primer F:aaggatccat gagtaaagaa agtggcaaat g

Reverse primer R:agtcgactca cagttggcta tacctaaagt cat

This primer is introduced BamH I restriction enzyme site to the target sequence upstream of amplification, and SalI restriction enzyme site, about 480bp are introduced in the downstream.

1.2 blood specimen DNA extraction

Sample source: in December, 2007 the thrombopenic purpura infant, man, 12 years old; Blood specimen is so kind as to give by The Fourth Military Medical University's Xijing pediatric hospital.

The blood specimen that drying tube is preserved centrifugal (2000～3000rpm * 5min) get supernatant adds 65 ℃ of fully digestion down of lysate, through 95 ℃ of Proteinase K deactivations, the supernatant-20 ℃ preservation of the centrifugal back of 15000rpm * 15min.

1.3 PCR reaction

To primer, 1.2 described blood specimen DNA are that template is carried out the PCR reaction with 1.1 described primers; The PCR reaction conditions is: 95 ℃ of pre-sex change 5min, enter circulation, and 93 ℃ of sex change 60s, the 55 ℃ of 90s that anneal, 72 ℃ of 1min of renaturation, totally 35 circulations, 72 ℃ are extended 10min after the loop ends.Amplified production carries out 1% agarose gel electrophoresis, presses the H.Q.﹠amp of U-GENE company; .Q the operation of gel recovery test kit II specification sheets is carried out, and ℃ preservation of the target sequence dna solution-20 of acquisition is standby.

1.4 construction of recombinant plasmid

To target sequence and plasmid pQE30 double digestion, enzyme is cut capable 1% agarose gel electrophoresis of product and is reclaimed by BamH I restriction enzyme site, Sal I restriction enzyme site, reclaims fragment T4DNA ligase enzyme construction recombination plasmid pQE30-VP1 distinct zones.

1.5 the evaluation of recombinant plasmid

Transformed into escherichia coli M15 competence will transform back M15 puts Amp+ in the sterile state lower berth dull and stereotyped cultivation of LB, and 8 single bacterium colonies of white of picking are inoculated in 37 ℃ of shakes of LB nutrient solution respectively and spend the night at random, extract plasmid.Plasmid is carried out BamHI and SalI double digestion, and enzyme is cut product and is carried out 1% agarose gel electrophoresis, and to transformed bacteria M15 order-checking (entrust TaKaRa, the precious biotechnology in Dalian company limited finishes).Enzyme is cut product 1% agarose gel electrophoresis, and figure is as shown in Figure 1 as a result: swimming lane 1 is a recombinant plasmid pQE30-VP1 distinct zones double digestion product, and swimming lane 2 is DNA marke; Visible clear band explanation recombinant plasmid pQE30-VP1 distinct zones successfully constructs swimming lane 1 in the 480bp place.Recombinant plasmid pQE30-VP1 distinct zones sequencing result passes through relatively as shown in Figure 3, and the sequence that obtains the VP1 distinct zones is shown in SEQID NO.1.

2 Recombinant Protein Expression, purifying

2.1 Recombinant Protein Expression

The M15 bacterium that will contain recombinant plasmid pQE30-VP1 is inoculated in by 1:100 that 37 ℃ of shakes spend the night in the LB nutrient solution that contains Amp+, and the bacterium liquid that spends the night is transferred by 1:50 and cultivated in the new LB nutrient solution that contains Amp+, surveys OD ₂₆₀Be 0.6, add IPTG and induce that 5500rpm collects thalline behind the 5h, ice STE, N,O-Diacetylmuramidase, 4% Sodium desoxycholate, 1M MgCl2, DNase1 cracking thalline, ultrasonic cracking once more.The centrifugal 10min of ultrasonic after product 10000rpm gets supernatant-70 ℃ preservation.

2.2 the purifying of recombinant protein

By Ni sepharose6 FF metal chelating column, source 30RPC reversed phase chromatography column purification albumen, Ni sepharose6 FF metal chelating column renaturation, the accurate purifying of Sephacryl 100 gel columns obtains purification of recombinant proteins B19-XA VP1 distinct zones albumen, and its sequence is shown in SEQ ID NO.2.

2.3 the evaluation of recombinant protein

Adopt SDS-PAGE, Western blot to identify recombinant protein, Western blot schemes, the visible clear band in the 22kDa place as shown in Figure 2, and recombinant protein successfully constructs.

3 recombinant protein pQE30-VP1 distinct zones immunization experiments

3.1 lymphocyte transformation experiment

With reference to " cell and molecular immunology experimental technique " (Jin Baiquan chief editor), select for use 6 the week age Balb/C mouse, isolated lymphocytes, experimental group adds the B19-XA VP1 distinct zones albumen behind the purifying, positive control adds PHA-P HA, and negative control does not add any material, cultivates 72 hours, add MTT and continue to cultivate after 4 hours, every hole adds dimethyl sulfoxide (DMSO) DMSO and ends.Spectrophotometer 570nm surveys the OD value, calculates stimulation index SI.

VP1 distinct zones albumen stimulating group SI=4.5, phytohaemagglutinin stimulating group SI=4.7, prompting VP1 distinct zones proteoplast external enwergy stimulates the T lymphocyte.

3.2 immunocompetence is measured

With reference to " cell and molecular immunology experimental technique ", select for use 6 age in week the Balb/C mouse, B19-XAVP1 distinct zones albumen adds with complete Freund's adjuvant or incomplete Freund's adjuvant immune animal, adopts the ELISA method to measure and produces antibody.The protein content of each immune mouse is 25ug, and immunity is 4 times altogether, produces antibody titer and reaches 1:8000.

The success that B19-XA VP1 distinct zones albumen stimulates the T lymphocyte and produces the immunization experiment of antibody, illustrate that VP1 unique region gene disclosed by the invention and recombinant protein B19-XA VP1 thereof provide the diagnostic antigen of new B19 virus, and can be used in the antibody of control B19 virus and the preparation of vaccine.

4 BP1 virus VP 1 distinct zones sequential analyses

Institute is obtained sequence typing NCBI Genbank website (http://www.ncbi.nlm.nih.gov), analyze by BLAST software, the result is shown in specification sheets 11-12 page or leaf, the most approaching with the EU478564.1 homology of delivering among the Genbank, but also have three nucleotide sites inconsistent (C203A), (A293G), (A422C).

The invention provides the nucleotide sequence of BP1 virus VP 1 distinct zones, made up B19 prokaryotic expression pQE30-VP1 distinct zones cloned plasmids, expressed and be purified into recombinant protein, externally can cause immune cell responses, produce high antibody of tiring; The diagnostic antigen of new B19 virus is provided, and has been used to prevent and treat the preparation of the antibody and the vaccine of B19 virus.

B19 virus VP1 distinct zones Nucleotide and aminoacid sequence table

＜110〉The Fourth Military Medical University of P.L.A

＜120〉a kind of B19 virus VP1 unique region gene

<160>1

<210>1

<211>480

<212>DNA

＜213〉synthetic

<400>1

atgagtaaag?aaagtggcaa?atggtgggaa?agtgatgata?aatttgctaa?agctgtgtat 60

cagcaatttg?tggaatttta?tgaaaaggtt?actggaacag?acttagagct?tattcaaata 120

ttaaaagatc?attataatat?ttctttagat?aatcccctag?aaaaccaatc?ctctctgttt 180

gacttagttg?ctcgtattaa?aaataacctt?aaaaactctc?cagacttata?tagtcatcat 240

tttcaaagtc?atggacggtt?atctgaccac?ccccatgcct?tatcatccag?tagcagtcat 300

gcagaaccta?gaggagaaaa?tgcagtatta?tctagtgaag?acttacacaa?gcctgggcaa 360

gttagcgtac?aactacccgg?tactacctat?gttgggcctg?gcaatgagct?acaagctggg 420

cccccgcaaa?gtgctgttga?cagtgctgca?aggattcatg?actttaggta?tagccaactg 480

<210>2

<211>160

<212>PRT

＜213〉synthetic

<400>2

Met?Ser?Lys?Glu?Ser?Gly?Lys?Trp?Trp?Glu?Ser?Asp?Asp?Lys?Phe?Ala?Lys?Ala?Val?Tyr

1 5 10 15 20

Gln?Gln?Phe?Val?Glu?Phe?Tyr?Glu?Lys?Val?Thr?G1y?Thr?Asp?Leu?Glu?Leu?Ile?Gln?Ile

21 25 30 35 40

Leu?Lys?Asp?His?Tyr?Asn?Ile?Ser?Leu?Asp?Asn?Pro?Leu?Glu?Asn?Gln?Ser?Ser?Leu?Phe

41 45 50 55 60

Asp?Leu?Val?Ala?Arg?11e?Lys?Asn?Asn?Leu?Lys?Asn?Ser?Pro?Asp?Leu?Tyr?Ser?His?His

61 65 70 75 80

Phe?Gln?Ser?His?Gly?Arg?Leu?Ser?Asp?His?Pr0?His?A1a?Leu?Ser?Ser?Ser?Ser?Ser?His

81 85 90 95 100

Ala?Glu?Pr0?Arg?Gly?Glu?Asn?Ala?Val?Leu?Ser?Ser?Glu?Asp?Leu?His?Lys?Pro?Gly?Gln

101 105 110 115 120

Val?Ser?Val?Gln?Leu?PrO?G1y?Thr?Thr?Tyr?Val?Gly?PrO?Gly?Asn?Glu?Leu?Gln?Ala?Gly

121 125 130 135 140

Pro?PrO?Gln?Ser?Ala?Val?Asp?Ser?Ala?Ala?Arg?I1e?His?Asp?Phe?Arg?Tyr?Ser?G1n?Leu

141 145 150 155 160

Claims (4)

1, a kind of gene of B19 virus VP1 distinct zones is characterized in that, its nucleotide sequence is shown in SEQID NO.1.

2, a kind of recombinant protein of B19 virus VP1 distinct zones is characterized in that, its aminoacid sequence is shown in SEQ ID NO.2.

3, the gene constructed prokaryotic expression carrier of the described B19 virus VP1 of a kind of claim 1 distinct zones is characterized in that, by BamH I, the Sal I restriction enzyme site structure prokaryotic expression carrier pQE30-VP1 distinct zones of plasmid pQE30.

4, the gene of the described B19 virus VP1 of claim 1 distinct zones is characterized in that, being applied to the B19 virus is the preparation of diagnostic antigen, antibody or vaccine of target spot.

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