CN1408851A - Non-coding region and P.M gene sequence and vector construction of long-47 measles virus - Google Patents

Abstract

The present invention provides coding sequences and structures of all the non-coding regions and P and M coding regions of Longchun-47 measles virus, protein amino acid sequence and structure of P and M coding regions, the use of the non-coding region sequences in constituting the vaccine virus carrier and the constituting process of the carrier. These data and method may be used in recombining multiple effect vaccine or gene treatment via utilizing the vaccine strain virus to constitute expression vector and may be also used in molecular biological diagnosis and reagent development for measles virus infection.

Description

Long-47 Measles virus non-coding regions and P, M gene order and vector construction

Invention field

The invention belongs to virusology and biology field, relate to discovery to all non-coding regions of a strain measles virus vaccines strain and P, M coding region sequence, exactly be all non-coding area sequences of controlling being used to of illustrating that a strain China successfully develops the attenuated live vaccine strain of measles, the aminoacid sequence of P, M coding region sequence and encoded protein matter thereof and their range of application.

Background of invention

Measles virus (Measles Virus, MV) being Paramyxoviridae Morbillivirus (morbillivirus) member, is a kind of sub-thread minus-stranded rna virus of non-segmented negative, also is one of virus that causes in the whole world hyperinfection disease, by respiratory tract spittle droplet, perhaps contact infection.Susceptible population almost reaches 100% to the sickness rate of measles, and most of the infected can recover fully, but minority can cause serious respiratory tract or central nervous system complication.

Long-47 vaccine strains are the vaccine strain of China's independent development, originate from isolating Leningrad-4 strain of nineteen fifty-seven Measles virus, passing 26 generations, human amniotic cell through human kidney cells passes and adapts to attenuation gradually after 47 generations and chick embryo fibroblast pass some generations and form (Rota, J.S., Wang, Z.D., Rota, P.A., and Bellini, W.J.1994.Comparision of sequences of theH, F, and N coding genes of measles virus vaccine strains.VirusResearch 31:317-330).China since nineteen sixty-five is brought into use Measles Vaccine, especially carried out planned immunization in 1987 after, measles control work has obtained major progress, security of Measles Vaccine and validity have obtained good proof.

Virus genomic research is just risen in the Protocols in Molecular Biology development seventies and after being used widely.Till the eighties in 19th century, the sub-thread minus-stranded rna virus many members' of family gene order is measured, thereby impelled the rise that utilizes minus-stranded rna virus cDNA to carry out the reverse genetics research of virus rescue, also caused utilizing it to study viral mechanism of causing a disease, persistent infection, vaccine attenuation mechanism and development new generation vaccine and structure virus expression carrier etc.Especially Bing Du non-coding area sequence (non-coding region between genome end and structure gene) has the cis regulatory elements function, can instruct transcribing and duplicating of downstream gene, has unusual meaning for making up virus expression carrier, have obvious advantage and utilize Measles virus to be built into expression vector: 1 compares with dna viral vector, the Measles virus rna replicon is without the DNA stage, so not can with cell chromosome generation recombination and integration, relatively safety; 2 compare with the positive chain RNA virus carrier, be difficult for taking place the virogene reorganization, thereby help keeping the stable of foreign gene, because the rna virus cdna reorganization depends on the mechanism that template is selected, and the minus-stranded rna virus genome mainly exists with ribonucleoprotein mixture (RNP) form, so recombination efficiency is much lower; 3, Measles virus is genomic effectively duplicates the propagation rule that must follow " the genome nucleotide number is 6 multiple ", thereby still the probability of survival is much smaller after genome generation insertion and the disappearance.4, with the Measles virus attenuated vaccine strain construction of expression vector of the safety of extensively being approved, the security of using in the body is higher.Therefore, the research of relevant Measles virus expression vector has very important significance.

Based on Measles virus as many advantages that carrier had, and the length of China-47 attenuated live vaccine is the traditional method development, its safe reliability is checked for a long time and is approved, but its genome non-coding area sequence and P, M coding region sequence are not illustrated as yet.The intellecture property that this can not protect the MV vaccine of China on the one hand from gene level has also limited on the other hand and has utilized this vaccine virus to make up transgenosis and expression vector.

Summary of the invention

Task of the present invention is to illustrate a kind of all non-coding region nucleotide sequences, viral P, M encoding histone region nucleotide sequence and aminoacid sequence thereof of long-47 strains of Measles virus attenuated live vaccine of safe, effective, widely used prevention measles.The present invention, by various combination, can directly instruct expression of exogenous gene and be provided at the site that is inserted with the function foreign gene in the viral genome by non-coding region, be used to make up and carry the carrier that foreign gene shifts and expresses, and this carrier also can be used for gene therapy.By research, disclose the mechanism that this sequence-directed downstream gene is transcribed, expressed to different non-coding area sequence functions.This invention can directly instruct utilization reverse genetic technology to grow-47 virus rescues on the cDNA level, can utilize mutating technology on purpose to change the viral genome structure and also obtain to have infective transformation virus, thereby greatly facilitate the duplicating of virus, propagation and Study of packaging, and can cause the appearance of novel attenuated live vaccine.Detailed Description Of The Invention

The present invention realizes by following technique means: the full gene of at first using long-47 vaccine strains of RT-PCR method segmentation amplification MV, be cloned into respectively and carry out dna sequence analysis in the plasmid vector, obtain all non-coding area sequences of this virus and P, M coding region sequence.

Produce for the chick embryo fibroblast primary cell culture through long-47 vaccine strains of the Measles virus of traditional biological method development, the present invention is from the cell extraction whole-cell rna of this vaccine virus infection, become cDNA with the random primer reverse transcription, and as template, with DNA polymerase chain reaction (DNA Polymerase ChainReaction, PCR) long-47 viral genome cDNA of amplification.6 sections overlapped fragments of coamplification cover whole length-47 viral genome.The gene of segmentation amplification is cloned on the plasmid vector respectively, and then zone to be measured carried out sequencing, obtain all seven sections non-coding area sequences of viral genome and its structure gene P, M coding region sequence, and derive P, M gene coding amino acid sequence (seeing below sequence table).

The present invention has illustrated all non-coding area sequences of Measles Vaccine,Live strain long-47 of China's development and P, M coding region sequence at home and abroad first, directly provide the insertion site and the inserted mode of the full genome carrier of Measles virus foreign gene to enable to duplicate, transcribe, pack and express with viral genome, thereby the present invention can directly cause the generation of Measles virus carrier (carrying single or multiple foreign genes), this carrier both had been expected to be used to make up the gene recombination polyvalent vaccine, also was expected to be used for gene therapy purpose.In view of the advantage of minus-stranded rna virus carrier, and can be for further studying the prospect that research platform is provided as pathogenesis, attenuation mechanism, widely used Measles virus attenuated vaccine strain has its incomparable advantage.The present invention can also directly instruct to make up only has the terminal regulating and controlling sequence of Measles virus to control transcription of foreign genes, express and duplicate and packaging system as cis regulatory elements.

Following examples are described in detail for the application of long all non-coding area sequences of-47 attenuated live vaccines of Measles virus and P, M coding region sequence, but do not mean that restriction content of the present invention.

Non-coding area sequence can be provided at the site of inserting foreign gene in the full genome of virus and is used to make up the vaccine virus expression vector between long-47 vaccine strain genes of embodiment 1 on the cDNA level, is used to make up gene recombination polyvalent vaccine and/or gene therapy.

Measles virus is Paramyxoviridae Morbillivirus (morbillivirus) member, it is a kind of sub-thread minus-stranded rna virus of non-segmented negative, genome is formed by the cistron arranged in series of six non-overlapping copies: 3 '-N-P-M-F-H-L-5 ', encode altogether 8 albumen: N, P/C/V, M, F, H, L, the zone is a non-coding area sequence between terminal and each structure gene of genome two.Long-47 Measles Vaccine strains almost between each structure gene non-coding region all to exist with conservative GAA trinucleotide be the gene termination signal and the gene start signal sequence (exception: be CCC trinucleotide sequence between H/L) on boundary, they are controlling termination that 3 ' end upstream protein coding gene transcribes and the polyadenylation and 5 ' of mRNA holds transcribing of downstream protein coding gene to restart.Because this characteristics that intergenic region has, gene similar to length-47 viral protein on the band of foreign gene two ends can be inserted in the viral genome behind the non-coding region at interval, this insertion protein gene just can the similar mode of viral protein gene be present in the viral genome like this, and is expressed and duplicate, be packaged in the progeny virus.As: between the P/M gene, insert foreign gene, can use conventional cloning process non-coding area sequence between the cDNA of the full gene of virus level is with P/M to repeat adjacent connection, the middle restriction enzyme site that is suitable for that keeps, then foreign gene DNA is inserted this restriction enzyme site, form recombinant cDNA structure, use methods known in the art (as vaccinia virus/t7 rna polymerase transient expression system) that it is transcribed into RNA behind the recombinant cDNA transfered cell as Figure 12.Under the condition that helper virus albumen N, P and L are provided, the recombinant RNA of transcribing out can form has the RNP that duplicates with transcriptional capability, forms to have the new filial generation recombinant virus of infection ability, thereby finishes the transfer and the expression of foreign gene.Foreign gene inserts other position of genome by that analogy, the foreign gene both sides of insertion are had and the similar non-coding area sequence with cis regulatory function (seeing Figure of description 1,2,3 respectively) in long-47 structure gene mRNA both sides, this also represents can insert in this viral genome the foreign gene more than 1, and obtains to express.

Long-47 vaccine strain genomes 3 ' terminal non-coding region of embodiment 2 Measles viruss and 5 ' end non-coding area sequence can be used for the defective virus particulate vector of construction expression foreign gene.The former comprises the start signal that viral anti-genome (Antigenome) duplicates by genome 3 ' terminal non-coding region, the start signal that the N protein mRNA is transcribed and in conjunction with proteic packaging signal of virus N etc.; The latter 5 ' end non-coding area sequence then comprises the start signal that viral genome (Genome) is duplicated, the termination signal that the L protein mRNA is transcribed and in conjunction with the proteic packaging signal of virus N etc.Will this section 3 ' terminal non-coding area sequence cDNA 3 ' hold routinely that cloning process connects downstream foreign gene initiator codon, and will this section 5 ' terminal non-coding area sequence cDNA 5 ' hold routinely that cloning process connects upstream foreign gene terminator codon, use methods known in the art (as in-vitro transcription or carry re-reading system in the body of rna polymerase gene) it to be transcribed into RNA and to import mammalian cell (as the Vero cell) then, this sequence can instruct the transcribing of " expressible gene " of insertion under the condition that viral L albumen (viral rna polymerase) exists, and under the condition that other albumen of virus exists, with N, P, the L protein binding forms the RNP mixture, and then duplicating and be packaged into the defective virus particle that carries foreign gene, foreign gene is expressed (seeing Figure of description 4) according to the similar mode of viral protein expression.

The coding region sequence and the amino acid sequence coded thereof of long-47 vaccine strain P of embodiment 3 Measles viruss and M gene can be used for making up gene recombinant antigens, are used for the serodiagnosis of viral infection of measles or the epidemiology survey after the vaccine inoculation.

According to the length of having measured-47 vaccine strain P and M protein gene sequence and amino acid sequence coded analytical results thereof, the complete genome sequence of the two be can choose respectively or the gene constructed protokaryon or the carrier for expression of eukaryon of major antigen epi-position comprised, in prokaryotic cell prokaryocyte and eukaryotic cell, express corresponding M and P albumen, be used for the serodiagnosis of viral infection of measles or the epidemiology survey after the vaccine inoculation behind the purifying.

All non-coding area sequences of embodiment 4 long-47 attenuated live vaccine strain virus, the amino acid sequence analysis of P, M coding region sequence and encoded protein matter thereof can be used for illustrating the attenuation mechanism of this vaccine strain and the mechanism of cell adapted cultivation.

All non-coding area sequences with length-47 attenuated live vaccine strain virus, the aminoacid sequence of P, M coding region sequence and encoded protein matter thereof compares with external street strain, vaccine strain viral genome corresponding position, illustrate the similarities and differences and the function corresponding change of growing between-47 attenuated live vaccines and external street strain and the vaccine strain from gene and protein level, can study the attenuation mechanism of vaccine, cell cultures coping mechanism and seek molecule marker between strong and weak strain, thus reference provided for the Molecule Epidemiology Investigation of Measles virus.

Nucleotide and aminoacid sequence table＜110〉Virology Inst., Chinese Academy of Preventive Medical Science＜120〉long-47 Measles virus non-coding regions and P, M gene order and vector construction＜140〉01141446.4＜141〉2001-9-20＜160〉11＜210〉1＜211〉107＜212〉RNA＜213〉Measles virus long-47 strains＜220〉＜223〉Measles virus long-47 vaccine strain genomes 3 ' terminal non-coding area sequences.＜400〉long-47 strains of 1accaaacaaa guuggguaag gauaguucaa ucaaugauca ucuucuagug cacuuaggau 60ucaagauccu auuaucaggg acaagagcag gauuagggau auccgag 107＜210〉2＜211〉121＜212〉RNA＜213〉Measles virus＜220〉＜223〉Measles virus long-47 pnca gene group N/P genes between non-coding area sequence.＜400〉2gugcgagagg ccgaggacca gaacaacauc cgccuacccu ccaucauugu uauaaaaaac 60uuaggaacca gguccacaca gccgccagcc caucaaccau ccacucccac gauuggagcc 120g 121＜210〉3＜211〉107＜212〉RNA＜213〉-47＜220〉＜223〉-47P/M。＜400〉long-47 strains of 3cuacagcuca acuuaccugc caaccccaug ccagucgacc caacuaguac aaccuaaauc 60cauuauaaaa aacuuaggag caaagugauu gccucccaag uuccaca 107＜210〉4＜211〉1012＜212〉RNA＜213〉Measles virus＜220〉＜223〉Measles virus long-47 vaccine strain genome M/F genes between non-coding area sequence.＜400〉4accguagugc ccagcaaugc ccgaaaacga ccccccucac aaugacagcc agaaggcccg 60gacaaaaaaa cccccuccga aagacuccac ggaccaagcg agaggccagc cagcagccga 120cggcaagcgc gaacaccagg cggccccagc acagaacagc cccgacacaa ggccaccacc 180agccacccca aucugcaucc uccucguggg acccccgagg accaaccccc aaggcugccc 240ccgauccaaa ccaccaaccg cauccccacc acccccggga aagaaacccc cagcaacugg 300aaggccccuc ccccucuccc ucaacacaag aacuccacaa ccgaaccgca caagcgaccg 360aggugaccca accgcaggca uccgacuccc uagacagauc cucucucccc ggcaaacuaa 420acaaaacuua gggccaagga acauacacac ccaacagaac ccagaccccg gcccacggcu 480ccgcgccccc aacccccgac aaccagaggg agcccccaac caaucccgca ggcucccccg 540gugcccacag gcagggacac caacccccga acagacccag cacccaacca ucgacaaucc 600aagacggggg ggccccccca aaaaaaggcc cccaggggcc gacagccagc accgcgagga 660agcccaccca ccccacacac gaccacggca accaaaccag aacccagacc acccugggcc 720accagcuccc agacucggcc aucaccccgc agaaaggaaa ggccacaacc cgcgcacccc 780agccccgauc cggcggggag ccacccaacc cgaaccagca cccaagagcg auccccgaag 840gacccccgaa ccgcaaagga caucaguacc ccacagccuc uccaaguccc ccggucuccu 900ccucuucucg aagggaccaa gagaucaauc caccacaccc gacgacacuc aacuccccac 960cccuaaagga gacaccggga aucccagaau caagacucau ccaaugucca uc 1012＜210〉5＜211〉160＜212〉RNA＜213〉-47＜220〉＜223〉-47F/H。＜400〉5uccucuacaa cucuugaaac acaaaugucc cacaagucuc cucuucguca ucaagcaacc 60accgcaccca gcaucaagcc caccugaaau uaucuccggc uucccucugg ccgaacaaua 120ucgguaguua auuaaaacuu agggugcaag aucauccaca 160＜210〉6＜211〉109＜212〉RNA＜213〉-47＜220〉＜223〉-47H/L。＜400〉6ggcugcuagu gaaccaaucu caugauguca cccagacauc aggcauaccc acuaguguga 60aauagacauc agaauuaaga aaaacgcagg guccaagugg uuccccguu 109＜210〉7＜211〉109＜212〉RNA＜213〉Measles virus long-47 strains＜220〉＜223〉Measles virus long-47 vaccine strain genomes 5 ' terminal non-coding area sequences.＜400〉7uugguugaac uccggaaccc uaauccugcc cuaggugguu aggcauuauu ugcaauauau 60uaaagaaaac uuugaaaaua cgaaguuucu auucccagcu uugucuggu 109＜210〉8＜211〉1524＜212〉RNA＜213〉Measles virus long-47 strains＜220〉＜223〉Measles virus long-47 vaccine strain genome p genes encoding region sequences.＜400〉8auggcagaag agcaggcacg ccaugucaaa aacggacugg aaugcauccg ggcucucaag 60gccgagccca ucggcucacu ggccaucgag gaagcuaugg cagcaugguc agaaauauca 120gacaacccag gacaggagcg agccaccugc agggaagaga aggcaggcag uucgggucuc 180agcaaaccau gccucucagc aauuggauca acugaaggcg gugcaccuug cauccgcggu 240cagggaccug gagagagcga ugacgacgcu gaaacuuugg gaaucccccc aagaaaucuc 300caggcaucaa gcacuggguu acagugucau uauguuuaug aucacagcgg ugaugcgguu 360aagggaaucc aagaugcuga cucuaucaug guucaaucag gccuugaugg ugauagcacc 420cucucaggag gagacaauga aucugaaaac agcgaugugg auauuggcga accugauacc 480gagggauaug cuaucacuga ccggggaucu gcucccaucu cuaugggguu cagggcuucu 540gauguugaaa cugcagaagg aggggagauc cacgagcucc ugagacucca auccagaggc 600aacaacuuuc cgaagcuugg gaaaacucuc aauguuccuc cgcccccgga ccccgguagg 660gccagcacuu ccgggacacc cauuaaaaag ggcacagacg cgagauuagc cucauuugga 720acggagaucg cgucuuuauu gacagguggu gcaacccaau gugcucgaaa gucacccucg 780gaaccaucag ggccaggugc accugcgggg aauguccccg agugugugag caaugccgca 840cugauacagg aguggacacc cgaaucuggu accacaaucu ccccgagauc ccagaauaau 900ggaaaagggg gagacuauua ugaugaugag cuguucucug auguccaaga uauuaaaaca 960gccuuggcca aaauacacga ggauaaucag aagauaaucu ccaagcuaga gucacugcug 1020uuauugaagg gagaaguuga gucaauuaag aagcagauca acaggcaaaa uaucagcaua 1080uccacccugg aaggacaccu cucaagcauc augaucgcca uuccuggacu ugggaaggau 1140cccaacgacc ccacugcaga ugucgaaauc aaucccgacu ugaaacccau cauaggcaga 1200gauucaggcc gagcacuggc cgaaguucuc aagaaacccg uugccagccg acaacuccaa 1260ggaaugacaa auggacggac caguuccaga ggacagcugc ugaaggaauu ucagcuaaag 1320ccgaucggga aaaagaugag cucagccguc ggguuuguuc cugacaccgg cccugcauca 1380cgcaguguaa uccgcuccau uauaaaaucc agccggcuag aggaggaucg gaagcguuac 1440cugaugacuc uccuugauga uaucaaagga gccaaugauc uugccaaguu ccaccagaug 1500cugaugaaga uaauaaugaa guag 1524＜210〉9＜211〉507＜212〉PRT＜213〉-47＜220〉＜223〉-47P。＜400〉9Met Ala Glu Glu Gln Ala Arg His Val Lys Asn Gly Leu Glu Cys5 10 15Ile Arg Ala Leu Lys Ala Glu Pro Ile Gly Ser Leu Ala Ile Glu20 25 30Glu Ala Met Ala Ala Trp Ser Glu Ile Ser Asp Asn Pro Gly Gln35 40 45Glu Arg Ala Thr Cys Arg Glu Glu Lys Ala Gly Ser Ser Gly Leu50 55 60Ser Lys Pro Cys Leu Ser Ala Ile Gly Ser Thr Glu Gly Gly Ala65 70 75Pro Cys Ile Arg Gly Gln Gly Pro Gly Glu Ser Asp Asp Asp Ala80 85 90Glu Thr Leu Gly Ile Pro Pro Arg Asn Leu Gln Ala Ser Ser Thr95 100 105Gly Leu Gln Cys His Tyr Val Tyr Asp His Ser Gly Asp Ala Val110 115 120Lys Gly Ile Gln Asp Ala Asp Ser Ile Met Val Gln Ser Gly Leu125 130 135Asp Gly Asp Ser Thr Leu Ser Gly Gly Asp Asn Glu Ser Glu Asn140 145 150Ser Asp Val Asp Ile Gly Glu Pro Asp Thr Glu Gly Tyr Ala Ile155 160 165Thr Asp Arg Gly Ser Ala Pro Ile Ser Met Gly Phe Arg Ala Ser170 175 180Asp Val Glu Thr Ala Glu Gly Gly Glu Ile His Glu Leu Leu Arg185 190 195Leu Gln Ser Arg Gly Asn Asn Phe Pro Lys Leu Gly Lys Thr Leu200 205 210Asn Val Pro Pro Pro Pro Asp Pro Gly Arg Ala Ser Thr Ser Gly215 220 225Thr Pro Ile Lys Lys Gly Thr Asp Ala Arg Leu Ala Ser Phe Gly230 235 240Thr Glu Ile Ala Ser Leu Leu Thr Gly Gly Ala Thr Gln Cys Ala245 250 255Arg Lys Ser Pro Ser Glu Pro Ser Gly Pro Gly Ala Pro Ala Gly260 265 270Asn Val Pro Glu Cys Val Ser Asn Ala Ala Leu Ile Gln Glu Trp275 280 285Thr Pro Glu Ser Gly Thr Thr Ile Ser Pro Arg Ser Gln Asn Asn290 295 300Gly Lys Gly Gly Asp Tyr Tyr Asp Asp Glu Leu Phe Ser Asp Val305 310 315Gln Asp Ile Lys Thr Ala Leu Ala Lys Ile His Glu Asp Asn Gln320 325 330Lys Ile Ile Ser Lys Leu Glu Ser Leu Leu Leu Leu Lys Gly Glu335 340 345Val Glu Ser Ile Lys Lys Gln Ile Asn Arg Gln Asn Ile Ser Ile350 355 360Ser Thr Leu Glu Gly His Leu Ser Ser Ile Met Ile Ala Ile Pro365 370 375Gly Leu Gly Lys Asp Pro Asn Asp Pro Thr Ala Asp Val Glu Ile380 385 390Asn Pro Asp Leu Lys Pro Ile Ile Gly Arg Asp Ser Gly Arg Ala395 400 405Leu Ala Glu Val Leu Lys Lys Pro Val Ala Ser Arg Gln Leu Gln410 415 420Gly Met Thr Asn Gly Arg Thr Ser Ser Arg Gly Gln Leu Leu Lys425 430 435Glu Phe Gln Leu Lys Pro Ile Gly Lys Lys Met Ser Ser Ala Val440 445 450Gly Phe Val Pro Asp Thr Gly Pro Ala Ser Arg Ser Val Ile Arg455 460 465Ser Ile Ile Lys Ser Ser Arg Leu Glu Glu Asp Arg Lys Arg Tyr470 475 480Leu Met Thr Leu Leu Asp Asp Ile Lys Gly Ala Asn Asp Leu Ala485 490 495Lys Phe His Gln Met Leu Met Lys Ile Ile Met Lys500 505＜210〉10＜211〉1008＜212〉RNA＜213〉-47＜220〉＜223〉-47M。＜400〉10augacagaga ucuacgacuu cgacaagucg gcaugggaca ucaaaggguc gaucgcuccg 60auacaaccca ccaccuacag ugauggcagg cuggugcccc aggucagagu cauagauccu 120ggucuaggcg acaggaagga ugaaugcuuu auguacaugu uucugcuggg gguuguugag 180gacagcgauc cccuagggcc uccaaucggg cgagcauuug ggucccugcc cuuagguguu 240ggcagaucca cagcaaagcc cgaaaaacuc cucaaagagg ccacugagcu ugacguaguu 300guuagacgua cagcagggcu caaugaaaaa cugguguucu gcaacaacac cccacuaacu 360cuccucacac cuuggagaaa gguccuaaca acagggagug ucuucaacgc aaaccaagug 420ugcaaugcgg cuaaucugau accgcucgau aacccgcaga gguuccgugu uguuuauaug 480agcaucaccc gucuuucgga uaacggguau uacaccguuc cuagaagaau gcuggaauuc 540agaucgguca augcaguggc cuucaaccug cuggugaccc uuaggauuga caaggcgaua 600ggcccuggga agaucaucga caauacagag caacuuccug aggcaacauu uaugguccac 660aucgggaacu ucaggagaaa gaagagugaa gucuacucug ccgauuauug caaaaugaaa 720aucgaaaaga ugggccuggu uuuugcacuu ggugggauag ggggcaccag ucuucacauu 780agaagcacag gcaaaaugag caagacucuc caugcacaac ucggguucaa gaagaccuua 840uguuacccgc ugauggauau caaugaagac cuuaaucgau uacucuggag gagcagaugc 900aagauaguaa gaauccaggc aguuuugcag ccaucaguuc cucaagaauu ccgcauuuac 960gacgacguga ucauaaauga ugaccaagga cuauucaaag uucuguag 1008＜210〉11＜211〉335＜212〉PRT＜213〉-47＜220〉＜223〉-47M。<400>11Met?Thr?Glu?Ile?Tyr?Asp?Phe?Asp?Lys?Ser?Ala?Trp?Asp?Ile?Lys5?10?15Gly?Ser?Ile?Ala?Pro?Ile?Gln?Pro?Thr?Thr?Tyr?Ser?Asp?Gly?Arg20?15?30Leu?Val?Pro?Gln?Val?Arg?Val?Ile?Asp?Pro?Gly?Leu?Gly?Asp?Arg35?40?45Lys?Asp?Glu?Cys?Phe?Met?Tyr?Met?Phe?Leu?Leu?Gly?Val?Val?Glu50?55?60Asp?Ser?Asp?Pro?Leu?Gly?Pro?Pro?Ile?Gly?Arg?Ala?Phe?Gly?Ser65?70?75Leu?Pro?Leu?Gly?Val?Gly?Arg?Ser?Thr?Ala?Lys?Pro?Glu?Lys?Leu80?85?90Leu?Lys?Glu?Ala?Thr?Glu?Leu?Asp?Val?Val?Val?Arg?Arg?Thr?Ala95?100?105Gly?Leu?Asn?Glu?Lys?Leu?Val?Phe?Cys?Asn?Asn?Thr?Pro?Leu?Thr105?110?120Leu?Leu?Thr?Pro?Trp?Arg?Lys?Val?Leu?Thr?Thr?Gly?Ser?Val?Phe125?130?135Asn?Ala?Asn?Gln?Val?Cys?Asn?Ala?Ala?Asn?Leu?Ile?Pro?Leu?Asp140?145?150Asn?Pro?Gln?Arg?Phe?Arg?Val?Val?Tyr?Met?Ser?Ile?Thr?Arg?Leu155?160?165Ser?Asp?Asn?Gly?Tyr?Tyr?Thr?Val?Pro?Arg?Arg?Met?Leu?Glu?Phe170?175?180Arg?Ser?Val?Asn?Ala?Val?Ala?Phe?Asn?Leu?Leu?Val?Thr?Leu?Arg185?190?195Ile?Asp?Lys?Ala?Ile?Gly?Pro?Gly?Lys?Ile?Ile?Asp?Asn?Thr?Glu200?205?210Gln?Leu?Pro?Glu?Ala?Thr?Phe?Met?Val?His?Ile?Gly?Asn?Phe?Arg2l5?220?225Arg?Lys?Lys?Ser?Glu?Val?Tyr?Ser?Ala?Asp?Tyr?Cys?Lys?Met?Lys230?235?240Ile?Glu?Lys?Met?Gly?Leu?Val?Phe?Ala?Leu?Gly?Gly?Ile?Gly?Gly245?250?255Thr?Ser?Leu?His?Ile?Arg?Ser?Thr?Gly?Lys?Met?Ser?Lys?Thr?Leu260?265?270His?Ala?Gln?Leu?Gly?Phe?Lys?Lys?Thr?Leu?Cys?Tyr?Pro?Leu?Met275?280?285Asp?Ile?Asn?Glu?Asp?Leu?Asn?Arg?Leu?Leu?Trp?Arg?Ser?Arg?Cys290?295?300Lys?Ile?Val?Arg?Ile?Gln?Ala?Val?Leu?Gln?Pro?Ser?Val?Pro?Gln305?310?315Glu?Phe?Arg?Ile?Tyr?Asp?Asp?Val?Ile?Ile?Asn?Asp?Asp?Gln?Gly320?325?330Leu?Phe?Lys?Val?Leu335

Description of drawings

Fig. 1 is long-47 vaccine strain genome cDNA structure iron of Measles virus, and shaded portion is a non-coding area sequence.

Fig. 2 is a non-coding region mode of connection between foreign gene and P/M gene, and to obtain recombinant viral genome, institute's shaded portion is non-coding area sequence between long-47 viral genome P/M protein genes of foreign gene insertion.

The foreign gene insert structure figure that Fig. 3 provides for long-47 vaccine strain virus vector genomes of Measles virus, dash area is a non-coding area sequence.

Fig. 4 is the mode of connection that only is connected with foreign gene by length-47 genome two terminal non-coding area sequences, and to make up the defective virus carrier, dash area is the terminal non-coding area sequence of genome.

Claims (10)

1. Measles virus grows-47 vaccine strains (Measles virus Changchun-47 attenuatedstrain) all non-coding area sequences of genome, comprise 7 discontinuous zones altogether, be that 6 structural gene codings separate out, it is respectively genome 3 ' terminal non-coding region, N/P, P/M, M/F, F/H, non-coding region and genome 5 ' terminal non-coding region between the H/L gene, it is characterized in that:(1) Measles virus-47 vaccine strain (Measles virus ChangChun-47 attenuatedstrain) base group 3 ' terminal non-coding area sequence is made up of 107 Nucleotide, is positioned at the 1-107 position Nucleotide of full-length gene group.3 '-5 ' direction RNA sequence is:noncoding region (totally 121 nucleotides) between long-47 vaccine strains (Measles virus Changchun-47 attenuatedstrain) the genome N/P gene of UGGUUUGUUUCAACCCAUUCCUAUCAAGUUAGUUACUAGUAGAAGAUCACGUGAAU CCUAAGUUCUAGGAUAAUAGUCCCUGUUCUCGUCCUAAUCCCUAUAGGCUC (2) measles virus; Be positioned at the 1686-1806 position nucleotides of full-length gene group; 3 '-5 ' direction RNA sequence is:CACGCUCUCCGGCUCCUGGUCUUGUUGUAGGCGGAUGGGAGGUAGUAACAAUA UUU UUUGAAUCCUUGGUCCAGGUGUGUCGGCGGUCGGGUAGUUGGUAGGUGAGGGUGCU AACCUCGGC, (3) is noncoding region, (totally 107 nucleotides) between long-47 vaccine strains, (Measles virus Changchun-47 attenuatedstrain) the genome P/M gene of measles virus; Be positioned at the 3331-3437 position nucleotides of full-length gene group; 3 '-5 ' direction RNA sequence is:GAUGUCGAGUUGAAUGGACGGUUGGGGUACGGUCAGCUGGGUUGAUCAUGUUG GAU UUAGGUAAUAUUUUUUGAAUCCUCGUUUCACUAACGGAGGGUUCAAGGUGU, (4) is noncoding region, (totally 1012 nucleotides) between long-47 vaccine strains, (Measles virus Changchun-47 attenuatedstrain) the genome M/F gene of measles virus; Be positioned at the 4446-5457 position nucleotides of full-length gene group; 3’-5’RNA：UGGCAUCACGGGUCGUUACGGGCUUUUGCUGGGGGGAGUGUUACUGUCGGUCUUCCGGGCCUGUUUUUUUGGGGGAGGCUUUCUGAGGUGCCUGGUUCGCUCUCCGGUCGGUCGUCGGCUGCCGUUCGCGCUUGUGGUCCGCCGGGGUCGUGUCUUGUCGGGGCUGUGUUCCGGUGGUGGUCGGUGGGGUUAGACGUAGGAGGAGCACCCUGGGGGCUCCUGGUUGGGGGUUCCGACGGGGGCUAGGUUUGGUGGUUGGCGUAGGGGUGGUGGGGGCCCUUUCUUUGGGGGUCGUUGACCUUCCGGGGAGGGGGAGAGGGAGUUGUGUUCUUGAGGUGUUGGCUUGGCGUGUUCGCUGGCUCCACUGGGUUGGCGUCCGUAGGCUGAGGGAUCUGUCUAGGAGAGAGGGGCCGUUUGAUUUGUUUUGAAUCCCGGUUCCUUGUAUGUGUGGGUUGUCUUGGGUCUGGGGCCGGGUGCCGAGGCGCGGGGGUUGGGGGCUGUUGGUCUCCCUCGGGGGUUGGUUAGGGCGUCCGAGGGGGCCACGGGUGUCCGUCCCUGUGGUUGGGGGCUUGUCUGGGUCGUGGGUUGGUAGCUGUUAGGUUCUGCCCCCCCGGGGGGGUUUUUUUCCGGGGGUCCCCGGCUGUCGGUCGUGGCGCUCCUUCGGGUGGGUGGGGUGUGUGCUGGUGCCGUUGGUUUGGUCUUGGGUCUGGUGGGACCCGGUGGUCGAGGGUCUGAGCCGGUAGUGGGGCGUCUUUCCUUUCCGGUGUUGGGCGCGUGGGGUCGGGGCUAGGCCGCCCCUCGGUGGGUUGGGCUUGGUCGUGGGUUCUCGCUAGGGGCUUCCUGGGGGCUUGGCGUUUCCUGUAGUCAUGGGGUGUCGGAGAGGUUCAGGGGGCCAGAGGAGGAGAAGAGCUUCCCUGGUUCUCUAGUUAGGUGGUGUGGGCUGCUGUGAGUUGAGGGGUGGGGAUUUCCUCUGUGGCCCUUAGGGUCUUAGUUCUGAGUAGGUUACAGGUAG ( 5 ) -47 ( Measles virus Changchun-47 attenuatedstrain ) F/H ( 160 ) ； Be positioned at the 7111-7270 position nucleotides of full-length gene group; 3 '-5 ' direction RNA sequence is:AGGAGAUGUUGAGAACUUUGUGUUUACAGGGUGUUCAGAGGAGAAGCAGUAGU UCG UUGGUGGCGUGGGUCGUAGUUCGGGUGGACUUUAAUAGAGGCCGAAGGGAGACCGG CUUGUUAUAGCCAUCAAUUAAUUUUGAAUCCCACGUUCUAGUAGGUGU, (6) be long-47 vaccine strains of measles virus, noncoding region between (Measles virus Changchun-47 attenuatedstrain) genome H/L gene, (totally 109 nucleotides); Be positioned at the 9125-9233 position nucleotides of full-length gene group; 3 '-5 ' direction RNA sequence is:long-47 vaccine strains, (Measles virus Changchun-47 attenuatedstrain) genome 5 ' the terminal non-coding area sequences, (totally 109 nucleotides) of CCGACGAUCACUUGGUUAGAGUACUACAGUGGGUCUGUAGUCCGUAUGGGUGAUCA CACUUUAUCUGUAGUCUUAAUUCUUUUUGCGUCCCAGGUUCACCAAGGGGCAA, (7) measles virus; Be positioned at the 15786-15894 position nucleotides of full-length gene group, 3 '-5 ' direction RNA sequence is:the 26S Proteasome Structure and Function of AACCAACUUGAGGCCUUGGGAUUAGGACGGGAUCCACCAAUCCGUAAUAAACGUUA UAUAAUUUCUUUUGAAACUUUUAUGCUUCAAAGAUAAGGGUCGAAACAGACCA (8) said gene.

2. measles virus grows-47 vaccine strains (Measles virus Changchun-47 attenuatedstrain) genome P gene coding region and M coding sequence; It is characterized in that:long-47 vaccine strains (Measles virus Changchun-47 attenuatedstrain) the genome P coding sequence of (1) measles virus is totally 1524 nucleotides; Be positioned at the 1807-3330 position nucleotides of full-length gene group; 3’-5’RNA：UACCGUCUUCUCGUCCGUGCGGUACAGUUUUUGCCUGACCUUACGUAGGCCCGAGAGUUCCGGCUCGGGUAGCCGAGUGACCGGUAGCUCCUUCGAUACCGUCGUACCAGUCUUUAUAGUCUGUUGGGUCCUGUCCUCGCUCGGUGGACGUCCCUUCUCUUCCGUCCGUCAAGCCCAGAGUCGUUUGGUACGGAGAGUCGUUAACCUAGUUGACUUCCGCCACGUGGAACGUAGGCGCCAGUCCCUGGACCUCUCUCGCUACUGCUGCGACUUUGAAACCCUUAGGGGGGUUCUUUAGAGGUCCGUAGUUCGUGACCCAAUGUCACAGUAAUACAAAUACUAGUGUCGCCACUACGCCAAUUCCCUUAGGUUCUACGACUGAGAUAGUACCAAGUUAGUCCGGAACUACCACUAUCGUGGGAGAGUCCUCCUCUGUUACUUAGACUUUUGUCGCUACACCUAUAACCGCUUGGACUAUGGCUCCCUAUACGAUAGUGACUGGCCCCUAGACGAGGGUAGAGAUACCCCAAGUCCCGAAGACUACAACUUUGACGUCUUCCUCCCCUCUAGGUGCUCGAGGACUCUGAGGUUAGGUCUCCGUUGUUGAAAGGCUUCGAACCCUUUUGAGAGUUACAAGGAGGCGGGGGCCUGGGGCCAUCCCGGUCGUGAAGGCCCUGUGGGUAAUUUUUCCCGUGUCUGCGCUCUAAUCGGAGUAAACCUUGCCUCUAGCGCAGAAAUAACUGUCCACCACGUUGGGUUACACGAGCUUUCAGUGGGAGCCUUGGUAGUCCCGGUCCACGUGGACGCCCCUUACAGGGGCUCACACACUCGUUACGGCGUGACUAUGUCCUCACCUGUGGGCUUAGACCAUGGUGUUAGAGGGGCUCUAGGGUCUUAUUACCUUUUCCCCCUCUGAUAAUACUACUACUCGACAAGAGACUACAGGUUCUAUAAUUUUGUCGGAACCGGUUUUAUGUGCUCCUAUUAGUCUUCUAUUAGAGGUUCGAUCUCAGUGACGACAAUAACUUCCCUCUUCAACUCAGUUAAUUCUUCGUCUAGUUGUCCGUUUUAUAGUCGUAUAGGUGGGACCUUCCUGUGGAGAGUUCGUAGUACUAGCGGUAAGGACCUGAACCCUUCCUAGGGUUGCUGGGGUGACGUCUACAGCUUUAGUUAGGGCUGAACUUUGGGUAGUAUCCGUCUCUAAGUCCGGCUCGUGACCGGCUUCAAGAGUUCUUUGGGCAACGGUCGGCUGUUGAGGUUCCUUACUGUUUACCUGCCUGGUCAAGGUCUCCUGUCGACGACUUCCUUAAAGUCGAUUUCGGCUAGCCCUUUUUCUACUCGAGUCGGCAGCCCAAACAAGGACUGUGGCCGGGACGUAGUGCGUCACAUUAGGCGAGGUAAUAUUUUAGGUCGGCCGAUCUCCUCCUAGCCUUCGCAAUGGACUACUGAGAGGAACUACUAUAGUUUCCUCGGUUACUAGAACGGUUCAAGGUGGUCUACGACUACUUCUAUUAUUACUUCAUC ( 2 ) -47 ( Measles virus Changchun-47 attenuatedstrain ) M1008； 3438-4445,3’-5’RNA：UACUGUCUCUAGAUGCUGAAGCUGUUCAGCCGUACCCUGUAGUUUCCCAGCUAGCGAGGCUAUGUUGGGUGGUGGAUGUCACUACCGUCCGACCACGGGGUCCAGUCUCAGUAUCUAGGACCAGAUCCGCUGUCCUUCCUACUUACGAAAUACAUGUACAAAGACGACCCCCAACAACUCCUGUCGCUAGGGGAUCCCGGAGGUUAGCCCGCUCGUAAACCCAGGGACGGGAAUCCACAACCGUCUAGGUGUCGUUUCGGGCUUUUUGAGGAGUUUCUCCGGUGACUCGAACUGCAUCAACAAUCUGCAUGUCGUCCCGAGUUACUUUUUGACCACAAGACGUUGUUGUGGGGUGAUUGAGAGGAGUGUGGAACCUCUUUCCAGGAUUGUUGUCCCUCACAGAAGUUGCGUUUGGUUCACACGUUACGCCGAUUAGACUAUGGCGAGCUAUUGGGCGUCUCCAAGGCACAACAAAUAUACUCGUAGUGGGCAGAAAGCCUAUUGCCCAUAAUGUGGCAAGGAUCUUCUUACGACCUUAAGUCUAGCCAGUUACGUCACCGGAAGUUGGACGACCACUGGGAAUCCUAACUGUUCCGCUAUCCGGGACCCUUCUAGUAGCUGUUAUGUCUCGUUGAAGGACUCCGUUGUAAAUACCAGGUGUAGCCCUUGAAGUCCUCUUUCUUCUCACUUCAGAUGAGACGGCUAAUAACGUUUUACUUUUAGCUUUUCUACCCGGACCAAAAACGUGAACCACCCUAUCCCCCGUGGUCAGAAGUGUAAUCUUCGUGUCCGUUUUACUCGUUCUGAGAGGUACGUGUUGAGCCCAAGUUCUUCUGGAAUACAAUGGGCGACUACCUAUAGUUACUUCUGGAAUUAGCUAAUGAGACCUCCUCGUCUACGUUCUAUCAUUCUUAGGUCCGUCAAAACGUCGGUAGUCAAGGAGUUCUUAAGGCGUAAAUGCUGCUGCACUAGUAUUUACUACUGGUUCCUGAUAAGUUUCAAGACAUC ( 3 ) 。

3. according to claim 2, long-47 vaccine strain P and the M gene corresponding structure albumen of encoding respectively, it is characterized in that: the albumen of (1) P genes encoding is made up of 507 amino acid, molecular weight is 53.788 kilodaltons, iso-electric point is 5.06, amino acid holds the C end to be from N: the albumen of MAEEQARHVKNGLECIRALKAEPIGSLAIEEAMAAWSEISDNPGQERATCREEKAG SSGLSKPCLSAIGSTEGGAPCIRGQGPGESDDDAETLGIPPRNLQASSTGLQCHYV YDHSGDAVKGIQDADSIMVQSGLDGDSTLSGGDNESENSDVDIGEPDTEGYAITDR GSAPISMGFRASDVETAEGGEIHELLRLQSRGNNFPKLGKTLNVPPPPDPGRASTS GTPIKKGTDARLASFGTEIASLLTGGATQCARKSPSEPSGPGAPAGNVPECVSNAA LIQEWTPESGTTISPRSQNNGKGGDYYDDELFSDVQDIKTALAKIHEDNQKIISKL ESLLLLKGEVESIKKQINRQNISISTLEGHLSSIMIAIPGLGKDPNDPTADVEINP DLKPIIGRDSGRALAEVLKKPVASRQLQGMTNGRTSSRGQLLKEFQLKPIGKKMSS AVGFVPDTGPASRSVIRSIIKSSRLEEDRKRYLMTLLDDIKGANDLAKFHQMLMKI IMK* (2) M genes encoding is made up of 335 amino acid, molecular weight is 37.650 kilodaltons, iso-electric point is 9.36, and amino acid holds the C end to be from N: the proteinic 26S Proteasome Structure and Function of above-mentioned all genes encodings of MTEIYDFDKSAWDIKGSIAPIQPTTYSDGRLVPQVRVIDPGLGDRKDECFMYMFLL GVVEDSDPLGPPIGRAFGSLPLGVGRSTAKPEKLLKEATELDVVVRRTAGLNEKLV FCNNTPLTLLTPWRKVLTTGSVFNANQVCNAANLIPLDNPQRFRVVYMSITRLSDN GYYTVPRRMLEFRSVNAVAFNLLVTLRIDKAIGPGKIIDNTEQLPEATFMVHIGNF RRKKSEVYSADYCKMKIEKMGLVFALGGIGGTSLHIRSTGKMSKTLHAQLGFKKTL CYPLMDINEDLNRLLWRSRCKIVRIQAVLQPSVPQEFRIYDDVIINDDQGLFKVL* (3).

4. according to the described feature of claim 1, the non-coding area sequence of long-47 vaccine strains of Measles virus can be used for making up long-47 vaccine virus expression vectors, is used to make recombination engineered vaccine or gene therapy purpose.

5. according to claim 1,2 and 4 described, it is characterized in that long-47 all non-coding area sequences, P and M gene order and restriction enzyme mapping thereof are used for the construction of recombinant virus carrier.

6. the purposes of claim 4, it is characterized in that: the foreign gene that is used for recombiant vaccine antigen purpose or gene therapy purpose is in non-coding region adjacent be connected of recombinant virus vector gene group with length-47.

7. according to the described feature of claim 1, terminal and 5 ' the art end non-coding area sequence of the genome 3 ' of long-47 vaccine strains of Measles virus can place the both sides of foreign gene, be used to control foreign gene duplicate, transcribe and pseudo-type packing with structure defective virus particulate vector.

8. according to the described feature of claim 4-7, contain the length-47 vaccine virus expression vector of a kind or more than one foreign genes.

9. according to claim 2 and 3 described features; the structure gene P and the M of long-47 vaccine strains of Measles virus; can be expressed by protokaryon or eukaryotic expression system; P expression of gene product or cloned plasmids also can provide this strain RNP mixture to form necessary P albumen, the subsidiary when being used for the recombinant virus rescue.

10. purposes according to Claim 8, P and M expression of gene product are used to set up the serological diagnostic method that measles infects.

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