阅读说明：本技术 一种用于诱导免疫的表位多肽与病毒载体组合 (Epitope polypeptide and virus vector combination for inducing immunity ) 是由 王革非 邓辉雄 谷李铭 李雁嫘 宋鑫利 王婷婷 何绚婷 代剑平 李蕊 于 2021-06-25 设计创作，主要内容包括：本发明公开了一种用于诱导免疫的表位多肽与病毒载体组合,属于生物技术领域；本发明提供了一种来源于多种肠道病毒的具有免疫保护生物学效应的表位多肽,将编码该表位多肽的基因负载于改造后的柯萨奇B3型病毒载体,经注射后可在细胞内成功表达外源蛋白；本发明将优选的表位多肽与病毒载体组合,相比单纯使用此重组表位多肽,可更加有效地诱导对多种病毒的免疫应答；表位多肽与病毒载体组合后,表位多肽编码的基因片段与病毒载体的相容性稳定,具备很好的遗传稳定性。(The invention discloses an epitope polypeptide and virus vector combination for inducing immunity, belonging to the technical field of biology; the invention provides an epitope polypeptide with immune protection biological effect from various enteroviruses, wherein a gene for coding the epitope polypeptide is loaded on a modified coxsackie B3 type virus vector, and exogenous protein can be successfully expressed in cells after injection; the invention combines the optimized epitope polypeptide with the virus vector, and compared with the simple use of the recombinant epitope polypeptide, the invention can more effectively induce the immune response to various viruses; after the epitope polypeptide is combined with the virus vector, the compatibility of the gene segment coded by the epitope polypeptide and the virus vector is stable, and the epitope polypeptide has good genetic stability.)

1. An epitope polypeptide, which is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

2. An epitope polypeptide, which is characterized by comprising a polypeptide which is composed of an amino acid sequence shown in SEQ ID No.1 through substitution and/or deletion and/or insertion of one or more amino acid residues and has the same function.

3. Gene encoding an epitope polypeptide as claimed in claim 1, characterized in that its nucleic acid sequence is as shown in SEQ ID No. 2.

4. A recombinant Coxsackie B3-type virus, which is characterized in that an epitope polypeptide as claimed in claim 1 or 2 is loaded by using an improved Coxsackie B3-type virus as a vector.

5. A vaccine composition comprising a recombinant coxsackie B3 type virus of claim 4 and a pharmaceutically acceptable carrier or adjuvant.

6. Use of the recombinant coxsackie B3 virus of claim 4 in the preparation of a medicament for preventing or treating intestinal diseases.

7. A pharmaceutical composition comprising the recombinant coxsackie B3 type virus of claim 4 and a pharmaceutically acceptable carrier or adjuvant.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an epitope polypeptide and virus vector combination for inducing immunity.

Background

Enteroviruses are a class of viruses belonging to the genus enterovirus of the family picornaviridae that can infect children and adults, and especially pose a significant threat to the health and public health of children. The viruses of enterovirus which threaten more greatly to children mainly comprise new enterovirus EV71 type, Coxsackie virus A16 type, Coxsackie virus A6 type and the like, the viruses can cause diseases such as hand-foot-and-mouth disease, herpangina and the like after infecting children, and the serious virus infection can cause central nervous system complications including encephalitis, encephalopathy and the like.

Because the main pathogenic viruses of the enteroviruses are various, and the immune cross reaction and protection of different enteroviruses are weak, the single-kind vaccine is difficult to play the immune protection role on multiple epidemic enteroviruses.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an epitope polypeptide for inducing immunity, which is combined with a virus vector, and the epitope polypeptide with immunoprotection biological effect is obtained by screening, optimizing and combining effective antigen epitopes and performing effective immunobiological analysis and computational biological verification.

The modified coxsackie B3 virus is used as a vector to load the epitope polypeptide, so that the obtained recombinant coxsackie B3 virus can generate effective immune response against a plurality of viruses such as a new enterovirus EV71 type, a coxsackie virus A16 type, a coxsackie virus B3 type and the like.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides an epitope polypeptide, and the amino acid sequence is shown in SEQ ID No. 1.

The invention also provides an epitope polypeptide, which comprises a polypeptide which is composed of an amino acid sequence shown in SEQ ID No.1 through substitution and/or deletion and/or insertion of one or more amino acid residues and has the same function.

The invention provides a gene for coding the epitope polypeptide as claimed in claim 1, and the nucleic acid sequence of the gene is shown as SEQ ID No. 2.

The invention provides a recombinant coxsackie B3 virus, which takes a modified coxsackie B3 virus as a vector and loads the epitope polypeptide.

The invention provides a vaccine composition, which comprises the recombinant coxsackie B3 virus and a pharmaceutically acceptable carrier or adjuvant.

The invention provides application of the recombinant coxsackie B3 virus in preparing a medicament for preventing or treating intestinal diseases.

The invention also provides a pharmaceutical composition, which comprises the recombinant coxsackie B3 virus and a pharmaceutically acceptable carrier or adjuvant.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides an epitope polypeptide with immune protection biological effect from a plurality of enteroviruses of a new enterovirus EV71 type, a coxsackievirus A16 type and a coxsackievirus A6 type, which simultaneously contains a molecular adjuvant, is different from the epitope polypeptide derived from traditional multi-viruses, the epitope polypeptide in the invention is obtained by screening, optimizing and combining effective antigen epitopes, carrying out effective immune biological analysis and computational biological verification, can well induce immune protection response aiming at common enteroviruses such as a plurality of viruses of a new enterovirus EV71 type, a coxsackievirus A16 type, a coxsackievirus A6 type, a coxsackievirus B3 type and the like, avoids ineffective immunity caused by the epitope with non-immune effect, balances cellular and humoral immunity, loads a gene for coding the epitope polypeptide on a modified coxsackievirus B3 type virus vector, after injection, the exogenous protein can be successfully expressed in the cells. The invention discloses a multivalent epitope vaccine capable of immunizing various enteroviruses, which is constructed by screening, identifying and docking analysis of dominant protective epitopes of various epidemic enteroviruses based on immune informatics, and analyzing the epitopes through computer cloning and immune simulation prediction.

The invention combines the optimized epitope polypeptide with the virus vector, and compared with the simple use of the recombinant epitope polypeptide, the invention can more effectively induce the immune response to various viruses; after the preferred epitope polypeptide is combined with a virus vector, the compatibility of a gene segment coded by the epitope polypeptide and the virus vector is stable, the defects of small loading exogenous gene segment, weak genetic stability and the like existing when the Coxsackie virus vector is used as a vector due to the characteristics of the Coxsackie virus vector are avoided, and the excellent genetic stability is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the results of electrophoresis of the expression products induced by the recombinant epitope polypeptide gene and the purified recombinant epitope polypeptide protein in example 1;

FIG. 2 shows the results of measuring the titer of specific antibodies of novel enteroviruses EV71, Coxsackie A16, Coxsackie A6 and Coxsackie B3 generated by ELISA-detected recombinant epitope polypeptides and recombinant Coxsackie B3-loaded viruses after mice are immunized with the recombinant epitope polypeptides.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

(1) Obtaining a gene fragment with a sequence number of SEQ ID No. 2:

the gene fragment SEQ ID No.2 of the epitope polypeptide shown by the coded SEQ ID No.1 is obtained by utilizing total gene synthesis or long primer splicing by a biosynthesis company, the sequence is optimized by a codon, and the sequence is as follows: 5'

ATGGCGAAACTGTCTACTGATGAACTGCTGGATGCATTCAAAGAGATGACCCTGCTGGAACTGTCCGATTTCGTTAAGAAATTCGAGGAAACCTTTGAAGTGACCGCAGCTGCGCCGGTTGCAGTTGCTGCTGCCGGCGCTGCGCCAGCGGGTGCAGCGGTTGAGGCAGCGGAAGAACAAAGCGAGTTCGACGTAATTCTGGAGGCAGCGGGCGACAAGAAAATTGGTGTAATCAAAGTAGTGCGTGAGATTGTGAGCGGTCTGGGCCTGAAAGAGGCGAAGGACCTGGTAGATGGTGCGCCGAAACCGCTGCTGGAGAAGGTTGCTAAGGAGGCGGCCGATGAAGCCAAAGCTAAACTGGAGGCAGCCGGCGCAACTGTTACCGTTAAAGAAGCGGCTGCGAAAGTTCCGCCGGGTGCGCCGAAGCCGGATAGCCGCGAGAGCCTGGCTTGGAAGAAACCAACCTTTGGCGAACACAAACAGGAGAAAGATCTGGAGTACGGCGCTGCCGCGTACTCCATGATCAATAACATTATCATCCGTGCCGCATACATCTCCAAATTCATTGACTGGCTGAAGGGTCCAGGCCCAGGCCCGGCTTCTGCATACCAGTGGTTTTATGATGGCTACCCGACCTTCGGTCCGGGTCCGGGCGTTCGCATCTACATGCGCATGAAACACGTTCGTGCTTGGATTCCGAAGAAATGGCAGACTGCAACTAACCCGTCTGTGTTCGTGAAGATGACTGATCCGAAGAAATATGACGGTTACCCGACCTTTGGTGAACACCTGCAGGCTAACGATCTGGCCGCTTATCGCGAGCAGGGTTGGATTATCCCGGAGGCCGCGTACGAAGTTACTTGGGAGAACGCTACTTTTGGCCCAGGCCCAGGCTGGGACTTCGGTCTGCAAAGCTCTGTGACCCTGGTGGTTCCGTGGGGTCCAGGTCCGGGTACTGCGGTTCAAGTGCTGCCAACCGCAGCAAACACCGAAGCAAGCAAGAAGCCGACTTTCGGTGAGCACAAACAGGCTACCAACCTGCAGTACGGTCAGAAGAAGGCGTCCATCACCACCACTGATTATGAGGGTGGTGTGCCGGCAAATCCGGCCGCATACATGATTAATAACATCATCATTCGTGCTGCCGCATACGCGACCGGTATCGTAACTATTTGGTATGGTCCGGGTCCGGGTCGTCCGATTCTGCGTACCGCGACTGTGCAGGGTCCGTCCCTGGATCATCATCATCATCATCACTGA-3', the two ends of the fragment can be introduced with specific enzyme cutting sites through PCR, or inserted into related expression plasmids or virus vectors through Gibson assembly, the sequence of the gene fragment can be modified according to the genetic rule, but the amino acid sequence of the coded epitope polypeptide is unchanged as shown in SEQ ID No.1, and specifically:

MAKLSTDELLDAFKEMTLLELSDFVKKFEETFEVTAAAPVAVAAAGAAPAGAAVEAAEEQSEFDVILEAAGDKKIGVIKVVREIVSGLGLKEAKDLVDGAPKPLLEKVAKEAADEAKAKLEAAGATVTVKEAAAKVPPGAPKPDSRESLAWKKPTFGEHKQEKDLEYGAAAYSMINNIIIRAAYISKFIDWLKGPGPGPASAYQWFYDGYPTFGPGPGVRIYMRMKHVRAWIPKKWQTATNPSVFVKMTDPKKYDGYPTFGEHLQANDLAAYREQGWIIPEAAYEVTWENATFGPGPGWDFGLQSSVTLVVPWGPGPGTAVQVLPTAANTEASKKPTFGEHKQATNLQYGQKKASITTTDYEGGVPANPAAYMINNIIIRAAAYATGIVTIWYGPGPGRPILRTATVQGPSLDHHHHHH；

(2) expression of recombinant epitope polypeptide genes: the epitope polypeptide gene fragment obtained above is introduced into a specific enzyme cutting site through PCR or is assembled and inserted into an expression vector through Gibson, as shown in figure 1, the epitope polypeptide gene fragment is inserted into a prokaryotic expression vector pET28a, and is transformed into escherichia coli BL21(DE3), and an expression band of the recombinant epitope polypeptide can appear at the expected molecular weight through SDS-PAGE electrophoresis after IPTG induction, and the result is shown in figure 1: the drawing is respectively provided with a protein Marker, epitope polypeptide after affinity chromatography purification, a waste protein liquid component 1 after affinity chromatography purification, a waste protein liquid component 2 after affinity chromatography purification, a waste protein liquid component 3 after affinity chromatography purification, escherichia coli containing recombinant epitope polypeptide expression plasmid after induction, before induction of escherichia coli containing recombinant epitope polypeptide expression plasmid and control escherichia coli from left to right; wherein the arrow position is marked as a recombinant epitope polypeptide protein band.

(3) Recombinant virus construction containing epitope polypeptide and virus vector combination

The Coxsackie virus B3 is modified by introducing specific enzyme cutting sites by PCR or Gibson assembly into a modified Coxsackie virus B3 expression Vector, transfecting a recombinant Vector into Vero cells or HeLa cells, and after 48 to 96 hours, the cells can be diseased, collecting the supernatant of the diseased cells, and obtaining a Coxsackie virus B3 epitope type recombinant Vector containing the Coxsackie virus and the Coxsackie virus B52 epitope in combination with the Coxsackie virus B52 according to the article 'Zeng J, Chen XX, Dai JP, Zhao XF, Xin G, Su Y,' Wang GF, Li KS. A patent substrate coated with Coxsackie virus B3 Vector: A Potential substrate for Viral Tracking and Gene Delivery, PLoS One,2013,8(12) 'e 83753'.

Effect verification

Example 1 immunization Induction experiment of recombinant epitope polypeptide obtained in step (2) and recombinant Coxsackie B3-type Virus obtained in step (3)

The recombinant epitope polypeptide and the recombinant coxsackie B3 virus are inoculated to mice in an injection mode. A control group, an epitope polypeptide inoculation group and a recombinant coxsackie B3 type virus (namely epitope polypeptide virus vector combination) group are arranged, and each group comprises 4 mice. The control group is not injected, the recombinant epitope polypeptide group is injected at 0 day and 14 day, each mouse is injected with 25 micrograms of recombinant epitope polypeptide, the recombinant coxsackie B3 type virus group is injected at 0 day and 14 day, and each mouse is injected with 10^4TCID 50. Serum of the mice was collected on 14 days of the second inoculation, and specific antibody titers of the new enterovirus EV71, coxsackievirus A16, coxsackievirus A6 and coxsackievirus B3 were detected in the serum. The specific antibody titers induced by the recombinant epitope polypeptide and the epitope polypeptide viral vector combination method against various enteroviruses are shown in fig. 2. The recombinant Coxsackie B3 virus prepared by the invention can induce and generate specific antibodies aiming at multiple viruses of new enterovirus EV71 type, Coxsackie virus A16 type, Coxsackie virus A6 type and Coxsackie virus B3 type, and the epitope polypeptide and the virus vector combination mode can induce higher specific antibody titer compared with the pure recombinant epitope polypeptide.

The above description is only for the preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention, the technical solution and the inventive concept of the present invention equivalent or change within the technical scope of the present invention.

Sequence listing

<110> Shantou university college of medicine

<120> an epitope polypeptide for inducing immunity in combination with a viral vector

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 419

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Lys Leu Ser Thr Asp Glu Leu Leu Asp Ala Phe Lys Glu Met

1 5 10 15

Thr Leu Leu Glu Leu Ser Asp Phe Val Lys Lys Phe Glu Glu Thr Phe

20 25 30

Glu Val Thr Ala Ala Ala Pro Val Ala Val Ala Ala Ala Gly Ala Ala

35 40 45

Pro Ala Gly Ala Ala Val Glu Ala Ala Glu Glu Gln Ser Glu Phe Asp

50 55 60

Val Ile Leu Glu Ala Ala Gly Asp Lys Lys Ile Gly Val Ile Lys Val

65 70 75 80

Val Arg Glu Ile Val Ser Gly Leu Gly Leu Lys Glu Ala Lys Asp Leu

85 90 95

Val Asp Gly Ala Pro Lys Pro Leu Leu Glu Lys Val Ala Lys Glu Ala

100 105 110

Ala Asp Glu Ala Lys Ala Lys Leu Glu Ala Ala Gly Ala Thr Val Thr

115 120 125

Val Lys Glu Ala Ala Ala Lys Val Pro Pro Gly Ala Pro Lys Pro Asp

130 135 140

Ser Arg Glu Ser Leu Ala Trp Lys Lys Pro Thr Phe Gly Glu His Lys

145 150 155 160

Gln Glu Lys Asp Leu Glu Tyr Gly Ala Ala Ala Tyr Ser Met Ile Asn

165 170 175

Asn Ile Ile Ile Arg Ala Ala Tyr Ile Ser Lys Phe Ile Asp Trp Leu

180 185 190

Lys Gly Pro Gly Pro Gly Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp

195 200 205

Gly Tyr Pro Thr Phe Gly Pro Gly Pro Gly Val Arg Ile Tyr Met Arg

210 215 220

Met Lys His Val Arg Ala Trp Ile Pro Lys Lys Trp Gln Thr Ala Thr

225 230 235 240

Asn Pro Ser Val Phe Val Lys Met Thr Asp Pro Lys Lys Tyr Asp Gly

245 250 255

Tyr Pro Thr Phe Gly Glu His Leu Gln Ala Asn Asp Leu Ala Ala Tyr

260 265 270

Arg Glu Gln Gly Trp Ile Ile Pro Glu Ala Ala Tyr Glu Val Thr Trp

275 280 285

Glu Asn Ala Thr Phe Gly Pro Gly Pro Gly Trp Asp Phe Gly Leu Gln

290 295 300

Ser Ser Val Thr Leu Val Val Pro Trp Gly Pro Gly Pro Gly Thr Ala

305 310 315 320

Val Gln Val Leu Pro Thr Ala Ala Asn Thr Glu Ala Ser Lys Lys Pro

325 330 335

Thr Phe Gly Glu His Lys Gln Ala Thr Asn Leu Gln Tyr Gly Gln Lys

340 345 350

Lys Ala Ser Ile Thr Thr Thr Asp Tyr Glu Gly Gly Val Pro Ala Asn

355 360 365

Pro Ala Ala Tyr Met Ile Asn Asn Ile Ile Ile Arg Ala Ala Ala Tyr

370 375 380

Ala Thr Gly Ile Val Thr Ile Trp Tyr Gly Pro Gly Pro Gly Arg Pro

385 390 395 400

Ile Leu Arg Thr Ala Thr Val Gln Gly Pro Ser Leu Asp His His His

405 410 415

His His His

<210> 2

<211> 1260

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcgaaac tgtctactga tgaactgctg gatgcattca aagagatgac cctgctggaa 60

ctgtccgatt tcgttaagaa attcgaggaa acctttgaag tgaccgcagc tgcgccggtt 120

gcagttgctg ctgccggcgc tgcgccagcg ggtgcagcgg ttgaggcagc ggaagaacaa 180

agcgagttcg acgtaattct ggaggcagcg ggcgacaaga aaattggtgt aatcaaagta 240

gtgcgtgaga ttgtgagcgg tctgggcctg aaagaggcga aggacctggt agatggtgcg 300

ccgaaaccgc tgctggagaa ggttgctaag gaggcggccg atgaagccaa agctaaactg 360

gaggcagccg gcgcaactgt taccgttaaa gaagcggctg cgaaagttcc gccgggtgcg 420

ccgaagccgg atagccgcga gagcctggct tggaagaaac caacctttgg cgaacacaaa 480

caggagaaag atctggagta cggcgctgcc gcgtactcca tgatcaataa cattatcatc 540

cgtgccgcat acatctccaa attcattgac tggctgaagg gtccaggccc aggcccggct 600

tctgcatacc agtggtttta tgatggctac ccgaccttcg gtccgggtcc gggcgttcgc 660

atctacatgc gcatgaaaca cgttcgtgct tggattccga agaaatggca gactgcaact 720

aacccgtctg tgttcgtgaa gatgactgat ccgaagaaat atgacggtta cccgaccttt 780

ggtgaacacc tgcaggctaa cgatctggcc gcttatcgcg agcagggttg gattatcccg 840

gaggccgcgt acgaagttac ttgggagaac gctacttttg gcccaggccc aggctgggac 900

ttcggtctgc aaagctctgt gaccctggtg gttccgtggg gtccaggtcc gggtactgcg 960

gttcaagtgc tgccaaccgc agcaaacacc gaagcaagca agaagccgac tttcggtgag 1020

cacaaacagg ctaccaacct gcagtacggt cagaagaagg cgtccatcac caccactgat 1080

tatgagggtg gtgtgccggc aaatccggcc gcatacatga ttaataacat catcattcgt 1140

gctgccgcat acgcgaccgg tatcgtaact atttggtatg gtccgggtcc gggtcgtccg 1200

attctgcgta ccgcgactgt gcagggtccg tccctggatc atcatcatca tcatcactga 1260