CN107384944B - Yeast-expressed Coxsackie virus A6 virus-like particle and application thereof - Google Patents

Abstract

The invention provides a yeast-expressed Coxsackie virus A6 virus-like particle and application thereof, and particularly provides a method for preparing the Coxsackie A6 virus-like particle, wherein a codon-optimized coding sequence of a P1 protein and a 3CD protein of the Coxsackie A6 virus is used in the method, the coding sequence is expressed in yeast cells, the VLPs can be automatically assembled to form the VLPs, the expression amount is high, the purification is easy, and the VLPs obtained by the purification have stronger immunogenicity.

Description

Yeast-expressed Coxsackie virus A6 virus-like particle and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to yeast-expressed Coxsackie virus A6 virus-like particles and application thereof.

Background

Hand-foot-and-mouth disease is a contact transmitted disease that is prevalent in the asia-pacific region. The main infections cause fever, herpes of hands, feet, mouth and buttocks, cough and other symptoms in children under five years of age, and a few children may have serious neurological symptoms and complications of the cardiopulmonary system in a short period, even death. However, no vaccine is currently available against hand-foot-and-mouth disease.

Since 2008, enterovirus 71 (EV 71) and coxsackievirus 16 (CA 16) have become two main pathogens of hand-foot-and-mouth disease due to their widely spread and epidemic, and research and development of vaccines are also directed against these two viruses. However, in recent years, reports on global epidemics of hand-foot-and-mouth disease caused by coxsackie CA6, especially epidemics in asia and europe, have been increasing, and have attracted widespread attention. The Coxsackie CA6 virus infection can cause typical hand-foot-and-mouth disease symptoms such as fever, herpes and the like, is accompanied by atypical hand-foot-and-mouth disease symptoms such as ulcer, scabbing and demethylation at a herpes outbreak, and can infect young children and seriously hurt adult people. Recent studies have reported that the capsid protein composition of Coxsackie CA6 virus particles is revealed, and that changes in the non-structural protein region (2A-3D) are a significant cause of atypical hand-foot-and-mouth disease symptoms caused by infection with this virus.

Taking clinical research reports of some provinces and cities in China as examples, in 2013, 60.3% of the hand-foot-and-mouth disease outbreaks of Guangdong province are caused by Coxsackie CA6 virus infection, and 66.9% of the cases of the hand-foot-and-mouth disease outbreaks of Jilin vinpocetine are also caused by the virus. These clinical research reports show that the prevalence of coxsackie CA6 is more and more extensive, and gradually becomes one of the main pathogens of the hand-foot-and-mouth disease outbreak, and moreover, the population immunized with EV71 or CA16 vaccine can still be infected by coxsackie CA6 virus, so that the vaccine development aiming at coxsackie CA6 is necessary, and the research and development of bivalent or multivalent vaccine can be laid.

Therefore, in order to effectively and specifically prevent coxsackie CA6 infection, the development of a vaccine aiming at coxsackie CA6 and application thereof are urgently needed in the field.

Disclosure of Invention

The invention aims to provide a coxsackie virus A6 virus-like particle expressed by yeast, a preparation method and application thereof.

In a first aspect of the invention, there is provided an isolated codon optimised polynucleotide encoding a coxsackie A6 virus P1 protein; and the polynucleotide is selected from the group consisting of:

(a) A polynucleotide with a sequence shown in SEQ ID NO. 3;

(b) Polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more) with the sequence shown in SEQ ID NO. 3;

(c) A polynucleotide complementary to any one of the polynucleotides of (a) - (c).

In a second aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the first aspect of the invention.

In another preferred embodiment, the expression vector further comprises a polynucleotide sequence encoding the coxsackie A6 virus 3CD protein.

In another preferred embodiment, the expression vector comprises a first expression cassette comprising the polynucleotide set forth in SEQ ID No.3 or a complement thereof; the second expression cassette comprises a polynucleotide shown in SEQ ID NO.8 or a complementary sequence thereof.

In another preferred embodiment, the expression vector is a recombinant baculovirus.

In another preferred embodiment, the first expression cassette further comprises a promoter located upstream of the polynucleotide shown in SEQ ID No.3, preferably the promoter is the PAOX1 promoter.

In another preferred embodiment, the second expression cassette further comprises a promoter located upstream of the polynucleotide shown in SEQ ID No.8, preferably the promoter is the PAOX1 promoter.

In a third aspect of the invention, there is provided a host cell comprising an expression vector according to the second aspect of the invention or having integrated into its genome a polynucleotide according to the first aspect of the invention.

In another preferred embodiment, the host cell is a yeast cell.

In a fourth aspect of the invention, there is provided a coxsackievirus A6 virus-like particle (VLP) expressed by a host cell according to the third aspect of the invention.

In another preferred embodiment, the virus-like particle comprises the polypeptides shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO. 6.

In another preferred embodiment, the virus-like particle consists of the polypeptides shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO. 6.

In a fifth aspect of the present invention, there is provided a method of preparing a coxsackievirus A6VLP comprising the steps of:

culturing the cell of the third aspect of the invention under conditions suitable for expression, thereby expressing the virus-like particle (VLP) of the fourth aspect of the invention; and isolating the Virus Like Particle (VLP).

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a virus-like particle (VLP) according to the fourth aspect of the invention, a polynucleotide according to the first aspect of the invention or an expression vector according to the second aspect of the invention or a host cell according to the third aspect of the invention, and a pharmaceutically acceptable carrier and/or adjuvant.

In another preferred embodiment, the pharmaceutical composition comprises a vaccine composition.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the adjuvant comprises alumina, saponin, quil A, muramyl dipeptide, mineral or vegetable oil, vesicle-based adjuvant, nonionic block copolymer or DEAE dextran, cytokines (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG).

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Drawings

FIG. 1 is a schematic diagram of P1 and 3CD protein (A) plasmid YCA6-003 of Pichia pastoris co-expressing CA6. TRP2-L and TRP2-R, upstream and downstream regions of TRP; PAOX1, AOX1 promoter; CYC1 TT, CYC1 transcription termination region; ADE2, encoding an aminoimidazole nucleotide carboxylase as a screening marker. And (B) screening high-expression strains. anti-CA 6VP0 was used as detection antibody. The yeast lysate of the empty vector transformation was used as a negative control (ctr). And (C) analyzing the expression state of the yeast clone by Western blot. Lane M: marker, lane C: yeast lysate from empty vector transformation, lanes 1-3: plasmid YCA6-003 is used to transform the lysates of strains 7, 12 and 15.

FIG. 2. Assembly of CA6 VLPs. The yeast lysate transformed by plasmid YCA6-003 is filtered by 10% -50%, and 12 layers are sequentially taken from top to bottom. (A) SDS-PAGE. (B) Western blotting, primary antibody: anti-CA 6VP 0. (C) Western blotting, primary antibody: anti-CA 6VP 1. (D) Western blotting, primary antibody: anti-CA 6VP 3. (E) Electron microscopy. Bar =100nm.

FIG. 3 shows the mouse immunization and antibody response. (A) SDS-PAGE analysis of CA6 VLPs against control antigen. (B) CA6VP0, VP1 and VP3 are mixed in equal proportion and coated on a plate for ELISA detection. Three-free four-week serum 1: and (5) diluting by 20. (C) the CA6VLP plates were used for ELISA detection. Three-free four-week serum 1:1000 dilution. (D) CA6 specific antibody titer detection. CA6 VLPs serve as envelope antigens. Each point represents a mouse and the horizontal line represents the geometric mean. The significant difference is as follows: * P <0.001.

Figure 4 anti-VLP sera conferred complete protection to mice. 7 day old ICR mice were injected intraperitoneally with 50. Mu.l of anti-VLP serum or control serum. 24 hours later, mice were injected intraperitoneally with (A, B) CA6/Gdula or (C, D) CA6/S0087b. Mice were recorded for 15 consecutive days of observation for (A, C) survival and (B, D) clinical symptoms. The clinical symptom score scale was as follows: 0, health; 1, bradykinesia; 2, ataxia; 3, paralysis; and 4, death.

Detailed Description

The inventor obtains a method for preparing the coxsackie A6 virus-like particle through extensive and intensive research, the coding sequences of the P1 protein and the 3CD protein of the coxsackie A6 virus optimized by a codon are used in the method, the coding sequences are expressed in yeast cells, the coding sequences can be automatically assembled to form VLPs, the expression amount is high, the purification is easy, and the VLPs obtained by the purification have stronger immunogenicity. On the basis of this, the present invention has been completed.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

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. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

The invention aims to research and develop a safe and efficient CA6 virus (coxsackie A6 virus) vaccine, which can protect organisms from being invaded by the virus. The P1 and 3CD proteins of CA6 are expressed by yeast, and specific polyclonal antibody detection shows that the P1 protein can be cut by 3CD to obtain capsid proteins VP0, VP1 and VP3, and the three structural proteins can be spontaneously assembled to form virus-like particles lacking nucleic acid. More importantly, anti-CA 6-VLP serum is successfully obtained after mice are immunized by the VLP vaccine, and the CA6-VLP vaccine can protect newborn mice from the attack of homologous virus CA6/Gdula and well protect the mice from the infection of heterologous virus CA6/S0087b. The CA6-VLP vaccine researched and developed by the invention can protect an organism from being invaded by a CA6 virus, and provides guarantee for the research and development of the hand-foot-and-mouth disease multivalent vaccine.

Hand, foot and mouth disease (Hand, foot and mouth disease, HFMD)

Hand-foot-and-mouth disease is a contagious disease that primarily infects children under five years of age, and is prevalent in asia pacific. Coxsackievirus type 6 (Coxsackievirus A6, CA 6) is one of the major causative agents of the disease, and reports of infection of children and adults by this virus have been increasing in recent years. However, there is no vaccine available against this virus.

In this study, correctly cut and assembled Virus-Like particles (VLPs) were obtained by co-expression of CA 6P 1 and 3CD using yeast cells. The results of the study show that the P1 protein can be correctly cleaved by 3CD to obtain the capsid proteins VP0, VP1 and VP3 which can spontaneously assemble to form complete VLPs. Mice immunized with the VLP vaccine can produce strong serum antibody responses, because the lack of a suitable cultured cell line for CA6 virus prevents the implementation of in vitro neutralization experiments, but passive and active protection in vivo is very effective. After seven-day-old newborn mice are subjected to intraperitoneal injection by using the anti-CA 6-VLP serum, the newborn mice are respectively attacked by using the original strain CA6/Gdula or the clinical isolate CA6/S0087b virus, and the result shows that the anti-VLP serum can protect the mice from being infected by homologous virus and heterologous virus. Meanwhile, the newborn suckling mice are selected to be immunized by intraperitoneal injection of CA6-VLP at the age of 1 day and 7 days, and are respectively attacked by original strain CA6/Gdula or clinical isolate CA6/S0087b virus at the age of 14 days, and the result shows that the CA6-VLP vaccine can also protect the mice from being infected by the two viruses.

Coxsackie virus type 6 (Coxsackie virus A6, CA 6)

Coxsackievirus type A6 (CA 6) is one of the main pathogens of Hand-foot-and-mouth disease (HFMD), the incidence of which is increasing in children and adults, and increasing CA6 infection poses a serious threat to public health, but no vaccine against CA6 is currently available.

Traditionally, inactivated vaccines and live attenuated vaccines are the most commonly used vaccine development strategies. Both methods require that the virus be able to propagate in the cell, however, CA6 is a Coxsackie virus that is difficult to culture. Moreover, CA6 does not replicate efficiently in the cells most commonly used in the production of both Vero and MRC-5 vaccines. Therefore, the development of CA6 vaccines by traditional methods would face significant challenges. In this study, the inventors explored the possibility of developing a CA6 vaccine based on the VLP strategy.

Since the CA6 virus is difficult to expand in cells, development of inactivated vaccines and live attenuated vaccines is limited. In this study, the present inventors developed a virus-like particle (VLP) based CA6 vaccine. The P1 and 3CD proteins of CA6 are co-expressed in Pichia pastoris to produce CA6-VLP, and after mice are immunized with the obtained VLP, CA 6-specific serum antibodies can be induced in the mice. In passive protection assays, antisera can protect mice from surviving a lethal challenge with CA6. The present invention successfully develops a CA6 vaccine based on the CA6VLP strategy.

Compared with other main pathogenic factors of hand-foot-and-mouth disease, namely CA16, CA10 and EV71, the Coxsackie A6 virus CA6 has significant differences, wherein the nucleotide homology is 76.4%, 81.5% and 73.8%, and the amino acid homology is 48.0%, 22.8% and 42.2%, so that the CA6 can be considered as a virus completely different from CA16, CA10 and EV 71.

The amino acid sequence of the coxsackie A6 virus P1 protein is as follows:

MGAQVSTEKSGSHETKNVATEGSTINFTNINYYKDSYAASASRQDFAQDPAKFTRPVLDTIREVAAPLQSPSVEACGYSDRVAQLTVGNSTITTQEAANIVLSYGEWPEYCPSTDATAVDKPTRPDVSVNRFYTLSTKSWKTESTGWYWKFPDVLNDTGVFGQNAQFHYLYRSGFCMHVQCNASKFHQGALLVAAIPEFVVAASSPATKPNGQGLYPDFAHTNPGKNGQEFRDPYVLDAGVPLSQALVYPHQWINLRTNNCATIIMPYVNALPFDSALNHSNFGLVVIPISPLKYCNGATTEVPITLTIAPLNSEFSGLRQAIKQGFPTELKPGTNQFLTTDDGTSPPILPGFEPTPLIHIPGEFTSLLDLCQIETILEVNNTTGTTGVSRLLIPVRAQNNVDQLCASFQVDPGRNGPWQSTMVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLIAYTPPGSAQPATREAAMLGTHIVWDFGLQSSVTLVIPWISNTHFRAVKTGGVYDYYATGIVTIWYQTNFVVPPDTPTEANIIALGAAQKNFTLKLCKDTDEIQQTAEYQNDPITNAVESAVSALADTTISRVTAANTAASTHSLGTGRVPALQAAETGASSNASDENLIETRCVMNRNGVNEASVEHFYSRAGLVGVVEVKDSGTNLDGYTVWPVDVMGFVQQRRKLELSTYMRFDAEFTFVSNLNDSTTPGMLLQYMYVPPGAPKPDSRKSYQWQTATNPSVFAKLSDPPPQVSVPFMSPATAYQWFYDGYPTFGEHKQATNLQYGQCPNNMMGHFAIRTVSESTTGKNVHVRVYMRIKHVRAWVPRPLRSQAYMVKNYPTYSQTITNTATDRASITTTDYEGGVPANPQRTS(SEQ ID NO.1)。

the wild-type polynucleotide sequence encoding the P1 protein of the coxsackie A6 virus is as follows:

ATGGGTGCCCAAGTTTCAACAGAAAAATCTGGGTCGCACGAGACAAAGAATGTAGCGACCGAAGGGTCTACTATCAACTTCACCAACATCAATTACTATAAGGATTCTTATGCAGCGTCAGCTAGTAGACAGGATTTTGCACAAGATCCCGCAAAGTTCACACGCCCTGTCTTGGATACCATCAGGGAGGTTGCAGCCCCGTTGCAATCCCCTTCTGTTGAGGCGTGCGGTTATAGTGACCGAGTCGCACAGTTGACTGTGGGCAACTCAACCATTACTACCCAAGAGGCAGCCAACATTGTGTTGAGCTACGGAGAGTGGCCAGAATATTGTCCCTCCACGGACGCTACAGCTGTGGACAAGCCTACTCGCCCTGACGTGTCAGTGAATAGGTTCTACACACTGTCAACTAAGAGTTGGAAGACAGAATCTACTGGCTGGTACTGGAAATTCCCTGATGTGCTAAATGACACAGGAGTATTCGGTCAAAACGCCCAATTCCACTACTTGTACCGCTCGGGTTTCTGCATGCACGTTCAGTGCAATGCAAGCAAGTTCCATCAGGGGGCCCTCTTAGTGGCTGCAATCCCCGAATTTGTGGTTGCTGCAAGCAGTCCTGCCACGAAGCCTAATGGACAAGGGTTGTACCCAGATTTCGCTCACACTAACCCGGGAAAAAATGGCCAAGAGTTTCGAGATCCTTATGTCTTGGATGCTGGTGTCCCCCTAAGTCAAGCACTGGTTTACCCCCATCAATGGATCAATCTACGAACTAATAACTGCGCGACCATTATCATGCCATATGTCAATGCGCTTCCATTTGATTCAGCGCTTAACCACTCAAATTTTGGATTGGTTGTGATCCCTATTAGCCCATTAAAATATTGTAATGGAGCTACCACAGAGGTGCCAATCACACTAACTATTGCCCCACTTAACTCGGAGTTTAGCGGCCTCCGACAAGCAATAAAACAAGGGTTTCCCACAGAGCTCAAGCCTGGGACCAATCAATTTCTTACAACTGATGACGGGACATCCCCACCAATACTGCCCGGTTTTGAACCAACTCCATTGATTCACATTCCTGGTGAGTTCACCTCTTTGTTAGATTTGTGTCAAATAGAAACCATACTAGAAGTCAATAATACCACTGGCACCACCGGGGTCAGTAGATTACTAATCCCCGTTCGAGCACAGAACAATGTGGACCAGTTGTGCGCATCATTCCAAGTAGACCCTGGGCGCAATGGCCCGTGGCAATCCACAATGGTCGGTCAGATCTGCAGGTATTACACTCAATGGTCAGGTTCCCTTAAGGTAACCTTTATGTTCACGGGTTCTTTTATGGCCACAGGGAAAATGCTGATAGCCTACACACCACCTGGTAGTGCTCAACCCGCTACAAGGGAAGCAGCAATGCTTGGGACTCATATAGTGTGGGATTTTGGTTTGCAATCATCGGTTACCCTAGTTATACCTTGGATTAGTAACACCCATTTTAGAGCAGTTAAGACTGGAGGGGTATACGACTACTACGCAACCGGGATCGTCACCATTTGGTACCAAACCAATTTCGTAGTGCCACCAGATACCCCCACTGAGGCTAATATTATAGCTCTTGGAGCAGCACAGAAAAACTTTACCCTAAAGTTGTGCAAGGACACTGACGAGATCCAGCAAACAGCAGAGTACCAAAATGATCCCATTACAAATGCAGTGGAAAGCGCTGTGAGCGCGCTTGCTGACACCACAATATCCCGGGTGACCGCAGCCAACACTGCAGCTAGCACTCACTCCCTGGGAACAGGGCGTGTACCAGCATTGCAAGCCGCAGAAACGGGAGCAAGCTCTAATGCTAGTGATGAGAACCTTATTGAGACTCGCTGTGTGATGAATCGAAACGGGGTTAATGAGGCGAGTGTGGAACACTTTTACTCTCGTGCAGGGCTGGTAGGAGTTGTGGAGGTGAAGGACTCGGGCACTAACCTGGATGGGTACACAGTTTGGCCTGTAGATGTGATGGGCTTCGTGCAACAGCGGCGCAAGCTAGAGCTGTCAACATACATGCGCTTTGATGCCGAGTTCACTTTTGTGTCCAACCTCAATGATAGCACGACGCCCGGGATGCTGCTGCAGTATATGTATGTACCACCAGGGGCTCCTAAGCCGGATAGCAGGAAATCATATCAATGGCAGACTGCTACTAACCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTATCTGTCCCGTTCATGTCGCCAGCAACAGCTTATCAGTGGTTTTATGATGGTTACCCTACATTTGGTGAGCACAAACAAGCTACCAATTTGCAATATGGGCAGTGTCCTAATAACATGATGGGCCATTTTGCCATCCGAACAGTCAGTGAATCTACCACCGGGAAAAATGTCCACGTTCGGGTGTACATGAGAATTAAGCACGTGAGAGCTTGGGTACCTAGACCCCTTCGATCCCAAGCTTATATGGTCAAAAACTACCCGACATACAGCCAAACAATAACTAACACTGCAACTGATCGTGCAAGTATAACCACCACGGATTATGAAGGCGGGGTACCAGCAAACCCACAAAGGACATCT(SEQ ID NO.2)。

in a preferred embodiment of the invention, the optimized coxsackie A6 virus P1 protein coding polynucleotide sequence is as follows:

ATGGGTGCTCAGGTGTCCACCGAGAAGTCCGGTTCCCACGAGACTAAGAACGTGGCCACCGAGGGTTCCACCATCAACTTCACCAACATCAACTACTACAAGGACTCCTACGCTGCTTCCGCTTCCCGTCAGGACTTCGCTCAGGACCCCGCTAAGTTCACCCGTCCCGTGCTGGACACTATCCGCGAAGTGGCTGCTCCCCTGCAGTCCCCATCTGTCGAGGCTTGCGGTTACTCCGACCGTGTGGCTCAGCTGACCGTGGGCAACTCTACCATCACCACCCAAGAGGCTGCTAACATCGTGCTGTCCTACGGCGAGTGGCCCGAGTACTGCCCTTCTACCGACGCTACCGCTGTGGACAAGCCCACCAGACCTGACGTGTCCGTGAACCGTTTCTACACCCTGTCCACCAAGTCCTGGAAGACCGAGTCCACCGGCTGGTACTGGAAGTTCCCCGACGTGCTGAACGACACCGGCGTGTTCGGACAGAACGCTCAGTTCCACTACCTGTACCGTTCCGGTTTCTGCATGCACGTCCAGTGCAACGCTTCCAAGTTCCACCAGGGTGCTCTGCTGGTGGCTGCTATCCCCGAGTTCGTCGTGGCTGCTTCATCCCCCGCTACCAAGCCTAACGGCCAGGGCCTGTACCCTGACTTCGCCCACACCAACCCTGGCAAGAACGGTCAAGAGTTCCGTGACCCCTACGTCCTGGACGCTGGTGTCCCTCTGTCTCAGGCTCTGGTGTACCCCCACCAGTGGATCAACCTGCGTACCAACAACTGCGCTACCATCATCATGCCCTACGTGAACGCTCTGCCCTTCGACTCCGCTCTGAACCACTCCAACTTCGGCCTGGTGGTCATCCCCATCTCCCCACTGAAGTACTGCAACGGTGCTACCACCGAGGTGCCCATCACCCTGACAATCGCTCCTCTGAACTCCGAGTTCTCCGGACTGCGTCAGGCTATCAAGCAGGGTTTCCCCACCGAGCTGAAGCCCGGTACTAACCAGTTCCTGACCACCGACGACGGCACCTCCCCACCTATCCTGCCTGGTTTCGAGCCCACCCCCCTGATCCACATCCCTGGCGAGTTCACCTCTCTGCTGGACCTGTGCCAGATCGAGACTATCCTGGAAGTGAACAACACCACCGGAACCACCGGTGTCTCCCGTCTGCTGATCCCTGTGCGTGCTCAGAACAACGTGGACCAGCTGTGCGCTAGCTTCCAGGTGGACCCCGGTCGTAACGGTCCTTGGCAGTCCACTATGGTCGGACAAATCTGCCGCTACTACACCCAGTGGAGCGGTTCCCTGAAAGTGACCTTCATGTTCACCGGTTCCTTCATGGCTACCGGCAAGATGCTGATCGCTTACACCCCCCCTGGTTCCGCTCAGCCCGCTACTCGTGAAGCTGCTATGCTGGGCACCCACATCGTGTGGGACTTCGGCTTGCAGTCCTCTGTGACCCTCGTGATCCCCTGGATCTCCAACACCCACTTCCGTGCTGTCAAGACCGGTGGCGTGTACGACTACTACGCTACCGGTATCGTGACCATCTGGTATCAGACCAACTTCGTGGTGCCCCCCGACACCCCTACCGAGGCTAACATCATCGCTCTGGGCGCTGCTCAGAAGAACTTCACCCTGAAGCTGTGCAAGGACACCGACGAGATCCAGCAGACCGCTGAGTACCAGAACGACCCCATCACCAACGCTGTCGAGTCCGCTGTGTCCGCTCTGGCTGACACCACCATCTCCCGTGTGACCGCTGCCAACACCGCTGCTTCTACCCACTCCCTGGGTACTGGTCGTGTGCCCGCTCTGCAGGCTGCTGAGACTGGTGCTTCCTCCAACGCCTCCGACGAGAACCTGATCGAAACCCGTTGCGTGATGAACCGTAACGGTGTCAACGAGGCTTCCGTCGAGCACTTCTACTCCCGCGCTGGACTCGTGGGCGTGGTGGAAGTTAAGGACTCCGGCACCAACCTGGACGGTTACACCGTCTGGCCCGTGGACGTGATGGGTTTCGTGCAGCAGCGTCGCAAGCTGGAACTGTCCACCTACATGCGTTTCGACGCTGAGTTCACTTTCGTGTCCAACCTGAACGACTCCACCACCCCCGGCATGCTGCTGCAGTACATGTACGTGCCCCCTGGTGCTCCCAAGCCCGACTCCAGAAAGTCCTACCAGTGGCAGACCGCCACCAACCCCTCCGTGTTCGCTAAGCTGTCCGACCCCCCACCACAGGTGTCCGTGCCTTTCATGTCCCCTGCTACCGCTTACCAGTGGTTCTACGACGGTTACCCCACCTTCGGCGAGCACAAGCAGGCTACCAACCTGCAGTACGGCCAGTGCCCCAACAACATGATGGGACACTTCGCTATCCGTACCGTGTCCGAGTCTACCACTGGAAAGAACGTCCACGTGCGTGTGTACATGCGTATCAAGCACGTGCGCGCTTGGGTGCCCCGTCCTCTGCGTTCCCAAGCTTACATGGTCAAGAACTACCCTACCTACTCCCAGACCATCACTAACACCGCTACCGACCGTGCTTCTATCACCACCACCGACTACGAGGGTGGTGTCCCCGCTAACCCTCAGAGGACCTCTTAA(SEQ ID NO.3)。

the Coxsackie A6 virus P1 protein is cut by 3CD protein to form VP0 protein, VP1 protein and VP3 protein, and the amino acid sequence of the VP0 protein is as follows:

MGAQVSTEKSGSHETKNVATEGSTINFTNINYYKDSYAASASRQDFAQDPAKFTRPVLDTIREVAAPLQSPSVEACGYSDRVAQLTVGNSTITTQEAANIVLSYGEWPEYCPSTDATAVDKPTRPDVSVNRFYTLSTKSWKTESTGWYWKFPDVLNDTGVFGQNAQFHYLYRSGFCMHVQCNASKFHQGALLVAAIPEFVVAASSPATKPNGQGLYPDFAHTNPGKNGQEFRDPYVLDAGVPLSQALVYPHQWINLRTNNCATIIMPYVNALPFDSALNHSNFGLVVIPISPLKYCNGATTEVPITLTIAPLNSEFSGLRQAIKQ(SEQ ID NO.4)；

the amino acid sequence of the VP1 protein is as follows:

NDPITNAVESAVSALADTTISRVTAANTAASTHSLGTGRVPALQAAETGASSNASDENLIETRCVMNRNGVNEASVEHFYSRAGLVGVVEVKDSGTNLDGYTVWPVDVMGFVQQRRKLELSTYMRFDAEFTFVSNLNDSTTPGMLLQYMYVPPGAPKPDSRKSYQWQTATNPSVFAKLSDPPPQVSVPFMSPATAYQWFYDGYPTFGEHKQATNLQYGQCPNNMMGHFAIRTVSESTTGKNVHVRVYMRIKHVRAWVPRPLRSQAYMVKNYPTYSQTITNTATDRASITTTDYEGGVPANPQRTS(SEQ ID NO.5)；

the amino acid sequence of the VP3 protein is as follows:

GFPTELKPGTNQFLTTDDGTSPPILPGFEPTPLIHIPGEFTSLLDLCQIETILEVNNTTGTTGVSRLLIPVRAQNNVDQLCASFQVDPGRNGPWQSTMVGQICRYYTQWSGSLKVTFMFTGSFMATGKMLIAYTPPGSAQPATREAAMLGTHIVWDFGLQSSVTLVIPWISNTHFRAVKTGGVYDYYATGIVTIWYQTNFVVPPDTPTEANIIALGAAQKNFTLKLCKDTDEIQQTAEYQ(SEQ ID NO.6)。

the amino acid sequence of the coxsackie A6 virus 3CD protein is as follows: (original strain CA6-Gdula-3 CD) GPSLDFALSLLRRNIRQAQTDQGHFTMLGVRDRLAILPRHSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLLTLDTNEKFRDITKFIPEAITGASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRSYFASEQGEIQWIKPNKETGRLNINGPTRTKLEPSVFHDVFEGNKEPAVLTSKDPRLEVNFEQALFSKYVGNTLHEPDEYVTQAALHYANQLKQLDINTSKMSMEEACYGTEYLEAIDLHTSAGYPYSALGIKKRDILDPATRDTSKMKLYMDKYGLDLPYSTYVKDELRSLDKIKKGKSRLIEASSLNDSVYLRMTFGHLYEVFHANPGTITGSAVGCNPDVFWSKLPILLPGSLFAFDYSGYDASLSPVWFRALELVLREIGYTEEAVSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIFNSMINNI IIRTLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTPADKSSCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRSLCLLAWHNGKEEYEKFVSTIRSVPIGKALAIPNFENLRRNWLELF (SEQ ID NO. 7).

The wild-type polynucleotide sequence encoding the coxsackie A6 virus 3CD protein is as follows: (original strain CA6-Gdula-3 CD)

GGACCTAGCTTGGACTTTGCTTTGTCTCTCCTGAGGCGTAACATCAGACAGGCGCAGACCGACCAGGGTCACTTCACCATGCTGGGCGTACGGGACCGCTTAGCTATCCTGCCACGCCACTCGCAACCAGGGAAAACCATCTGGATAGAACACAAATTGGTCAATGTATTAGATGCAGTTGAATTGGTGGATGAACAAGGTGTTAATTTAGAACTCACACTGCTGACCTTGGACACTAATGAGAAGTTTAGGGACATCACTAAGTTCATTCCAGAGGCAATCACTGGAGCGAGTGATGCAACTCTAGTTATCAACACTGAGCACATGCCCTCGATGTTTGTACCAGTAGGTGACGTTGTACAGTATGGGTTCTTGAATCTCAGTGGTAAACCTACTCACAGAACCATGATGTACAATTTCCCTACAAAGGCAGGACAATGTGGAGGGGTGGTCACCTCAGTTGGCAAGATCATTGGAATCCACATTGGCGGAAATGGACGTCAGGGCTTCTGCGCCGGCTTAAAGAGGAGCTACTTCGCCAGTGAACAAGGAGAAATCCAGTGGATAAAACCTAACAAAGAAACTGGGAGGCTGAATATTAATGGTCCAACTCGGACCAAATTGGAGCCCAGTGTATTCCATGATGTGTTCGAGGGCAACAAAGAGCCGGCGGTTTTGACCAGCAAGGATCCTAGGTTAGAGGTTAATTTTGAGCAAGCTCTGTTCTCTAAGTACGTGGGCAACACTCTACATGAACCTGATGAGTATGTGACACAAGCTGCCCTCCACTATGCAAATCAGCTGAAACAACTAGACATAAACACCAGCAAGATGAGCATGGAGGAGGCGTGCTATGGTACAGAATATTTGGAAGCAATAGACCTGCATACTAGTGCTGGGTACCCTTATAGCGCCCTGGGTATTAAGAAGAGAGACATTCTCGATCCAGCTACCAGAGACACTTCCAAGATGAAATTATACATGGACAAGTATGGACTGGATTTGCCCTACTCCACTTATGTAAAGGATGAGCTTAGATCTCTAGACAAAATTAAGAAAGGAAAGTCTCGCTTAATTGAGGCCAGCAGCCTAAATGACTCTGTCTACCTTAGAATGACTTTTGGTCATCTATATGAGGTGTTTCACGCCAACCCGGGAACCATAACTGGATCTGCAGTCGGGTGTAATCCTGATGTGTTCTGGAGTAAGCTGCCAATCTTACTGCCGGGCTCGCTCTTTGCATTTGACTACTCAGGATATGATGCAAGCCTTAGTCCTGTATGGTTTAGAGCTCTAGAGTTGGTTCTGCGGGAGATCGGTTACACGGAGGAGGCTGTGTCACTCATAGAAGGAATTAACCACACTCACCACGTGTACCGGAACAAGACATACTGTGTTCTTGGTGGGATGCCCTCAGGTTGCTCTGGTACTTCCATTTTCAATTCCATGATTAACAACATAATCATCAGAACCCTCTTGATTAAAACGTTCAAAGGTATAGACTTAGATGAATTGAACATGGTGGCCTACGGGGATGATGTGTTGGCTAGCTACCCATTTCCCATTGATTGCTTGGAATTGGCAAAAACTGGCAAGGAGTACGGATTGACCATGACTCCTGCCGACAAATCATCCTGTTTCAATGAAGTCACCTGGGAGAATGCAACTTTCTTAAAACGGGGTTTCTTACCAGATCATCAGTTTCCTTTTCTGATCCATCCCACCATGCCCATGAGGGAAATCCACGAGTCCATTCGCTGGACCAAGGATGCTCGTAATACTCAGGACCACGTGCGCTCCCTTTGTTTGCTGGCATGGCACAATGGAAAAGAGGAATATGAGAAATTTGTGAGCACAATTAGATCAGTTCCCATTGGAAAAGCTTTGGCAATACCAAATTTTGAGAACTTGAGAAGAAATTGGCTCGAACTATTTTAA(SEQ ID NO.8)。

Genetically engineered cell

The invention provides a gene engineering cell (host cell), which is a eukaryotic cell, and has an expression cassette of coxsackie A6 virus P1 protein and an expression cassette of 3CD protein integrated in the genome of the cell; or the cell contains an expression vector which contains an expression cassette of Coxsackie A6 virus P1 protein and an expression cassette of 3CD protein;

and the genetically engineered cell expresses a coxsackie A6 virus P1 protein and a 3CD protein intracellularly, and the P1 protein is cleaved by the 3CD protein to form capsid proteins VP0 protein, VP1 protein and VP3 protein, and the VP0 protein, VP1 protein and VP3 protein form virus-like particles (VLP) inside the genetically engineered cell.

In another preferred embodiment, the cell is a yeast cell.

In another preferred embodiment, the expression cassette of the coxsackie A6 virus P1 protein comprises the following elements operably linked 5 'to 3': a promoter, an initiation codon, an ORF sequence of a Coxsackie A6 virus P1 protein and a stop codon.

In another preferred embodiment, the expression cassette of the coxsackie A6 virus 3CD protein comprises the following elements operably linked 5 'to 3': a promoter, an initiation codon, an ORF sequence of a Coxsackie A6 virus 3CD protein and a stop codon.

In the present invention, the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

Compositions and methods of administration

The present invention also provides a composition comprising: (i) A recombinant virus-like particle (VLP) of the invention or a polynucleotide of the invention that can encode a recombinant virus-like particle, and (i i) a pharmaceutically or immunologically acceptable excipient or adjuvant.

In the present invention, the term "comprising" means that various ingredients can be applied or present together in the composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

The compositions of the present invention include pharmaceutical compositions and vaccine compositions.

The compositions of the invention may be monovalent (comprising only one recombinant virus-like particle or polynucleotide) or multivalent (comprising a plurality of recombinant virus-like particles or polynucleotides).

The pharmaceutical or vaccine compositions of the present invention may be prepared in a variety of conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.

(1) Pharmaceutical composition

The pharmaceutical composition of the present invention comprises (or contains) a therapeutically effective amount of the recombinant virus-like particle or polynucleotide of the present invention.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The effect can be detected, for example, by antigen levels. Therapeutic effects also include reduction of physiological symptoms. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given situation, routine experimentation may be used to determine the effective amount.

For the purposes of the present invention, an effective dose is about 0.001 mg/kg to 1000 mg/kg, preferably about 0.01 mg/kg to 100 mg/kg, of recombinant virus-like particles administered to a subject.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent (e.g., a recombinant virus-like particle of the invention). The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acid (polylactic acid), polyglycolic acid and the like. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. Co., n.j.1991).

Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, the compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; it can also be prepared into solid forms suitable for preparing solutions or suspensions, liquid vehicles before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.

(ii) Vaccine composition

The vaccine (composition) of the invention may be prophylactic (i.e. to prevent disease) or therapeutic (i.e. to treat disease after disease).

These vaccines comprise an immunizing antigen (including the recombinant virus-like particles of the invention), and are typically combined with "pharmaceutically acceptable carriers" which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants"). Alternatively, the antigen may be conjugated to a bacterial toxoid such as a toxoid from a pathogen such as diphtheria, tetanus, cholera, helicobacter pylori, and the like.

Preferred adjuvants to enhance the effect of the immunological composition include, but are not limited to: (1) Aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) Oil-in-water emulsion formulations such as (a) MF59 (see WO 90/14837), (b) SAF, and (c) Ribi Adjuvant System (RAS) (Ribi Immunochem, hamilton, MT), (3) saponin adjuvant; (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) Cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon), macrophage colony stimulating factor (M-CFS), tumor Necrosis Factor (TNF), etc.; (6) Detoxified variants of bacterial ADP-ribosylating toxins (e.g., heat labile toxin LT of e.coli); and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition.

Vaccine compositions, including immunogenic compositions (e.g., which may include an antigen, a pharmaceutically acceptable carrier, and an adjuvant), typically contain diluents such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.

More particularly, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as the other desired components described above. An "immunologically effective amount" refers to an amount that is therapeutically or prophylactically effective for administration to an individual as part of a single dose or a continuous dose. The amount will depend upon the health and physiological condition of the individual being treated, the class of individual being treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. It is expected that the amount will be within a relatively wide range and can be determined by routine experimentation.

Typically, the vaccine composition or immunogenic composition can be prepared as an injectable formulation, such as a liquid solution or suspension; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

Furthermore, the vaccine composition of the present invention may be a monovalent or multivalent vaccine.

(iii) Route of administration and dosage

Once the composition of the invention is formulated, it can be administered directly to the subject. The subject to be treated may be a mammal, especially a human.

When used as a vaccine, the recombinant virus-like particles of the present invention can be administered directly to an individual by known methods. These vaccines are typically administered using the same route of administration as conventional vaccines and/or mimicking the route of infection by pathogens.

Routes of administration of the pharmaceutical or vaccine compositions of the invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral, or other parenteral routes of administration. If desired, the routes of administration may be combined, or adjusted according to the disease condition. The vaccine composition may be administered in a single dose or in multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.

The recombinant virus-like particle vaccine should be administered in an "effective amount", i.e., an amount of recombinant virus-like particle sufficient to elicit an immune response in the route of administration chosen, effective to promote protection of the host against the associated disease.

Representative diseases include (but are not limited to): rabbit hemorrhagic disease virus infection, etc.

The amount of recombinant virus-like particles selected in each vaccine dose is based on the amount that elicits an immunoprotective response without significant side effects. Typically, each dose of vaccine is sufficient to contain about 1. Mu.g to 1000mg, preferably 1. Mu.g to 100mg, more preferably 10. Mu.g to 50mg of protein after infection of the host cell. Standard research methods including observing antibody titers and other responses in a subject can be used to determine the optimal amount of a particular vaccine. The need for booster doses can be determined by monitoring the level of immunity provided by the vaccine. After the antibody titer in serum is assessed, booster doses of immunization may be selected. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention.

The preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.

In addition, the vaccines of the present invention may be administered in conjunction with other immunomodulators, or with other therapeutic agents.

The main advantages of the invention are:

(1) The novel vaccine based on the Coxsackie A6 virus VLP is disclosed for the first time, the organisms can be protected from the infection of the CA6 virus more safely and efficiently, meanwhile, cross protection does not exist for main pathogenetic agents of the hand-foot-and-mouth disease such as CA16, CA10 and EV71, the CA6 harmfulness is increased, the development of the CA6 vaccine lays a foundation for broad-spectrum multivalent vaccines of the hand-foot-and-mouth disease, and the protection range of the hand-foot-and-mouth disease vaccine is expanded.

(2) The vector containing the Coxsackie A6 virus P1 protein expression cassette and the 3CD protein expression cassette constructed by the invention can successfully express the P1 protein and the 3CD protein of the Coxsackie A6 virus in a genetically engineered cell, the P1 protein is cut by the 3CD protein to form capsid proteins VP0 protein, VP1 protein and VP3 protein, and the VP0 protein, the VP1 protein and the VP3 protein can independently form virus-like particles (VLP) in the genetically engineered cell.

(3) The invention uses the P1 protein codon sequence of the Coxsackie A6 virus which is optimized for a plurality of times to construct the VLP expression vector, can efficiently express in yeast cells, and obviously improves the VLP yield. The 3CD protein plays an important role in the VLP forming process, the invention optimizes the 3CD protein codon sequence, and the result shows that the expression yield of the VLP cannot be obviously improved, so the wild type 3CD protein coding gene is still adopted in the invention.

(4) At present, the amplification effect of the CA6 virus on Vero and other cells is poor, and a recent study finds that the amplification titer of the CA6 virus on RD cells only reaches 10 ⁷ TCID50/mL, the yield of the virus is lower, the neutralization titer to the CA6 virus of the strain is lower, and the development of the inactivated vaccine of CA6 is greatly limited by a plurality of factors, and the application develops the novel CA6 vaccine based on the genetic engineering strategy, has obvious advantages in the aspects of virus yield, immunogenicity and the like, and breaks through the limitation bottleneck of the development of the inactivated vaccine of CA6.

The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Peitang, eds., beijing: scientific Press, 2002, USA, sambrook, et al, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

Materials and methods

1.1 strains and antibodies

Two strains of CA6 virus used in the experiment, CA6/Gdula (GenBank ID: AY 421764.1), were purchased from American strain collection center (ATCC # VR-165), and clinical strain CA6/S0087b (GenBank ID: KT 183533.1) was given by the centers for disease control and diagnosis of Shanghai Pasteur, china academy of sciences. The VP0 protein of CA6 is expressed in colibacillus, and after rabbit is immunized with the protein, the CA6VP0 polyclonal antibody is obtained, and by using the same method, the VP1 and VP3 proteins of CA6 are expressed and produced, and after mice are immunized respectively, the CA6VP1 polyclonal antibody and CA6VP3 polyclonal antibody are obtained.

1.2 vector construction

The cDNA gene of CA6/Gdula was obtained by reverse transcription). Then, a 3CD fragment was amplified using cDNA as a template and cloned into pPink-HC (Invitrogen) to produce plasmid YCA6-3CD. The P1 gene (SEQ ID NO. 1) of the optimized clinical strain SZC173/13 (GenBank ID: KF 682362.1) was synthesized in Kinsley, shanghai, china, cloned into pPink-HC (Invitrogen) to produce plasmid YCA6-P1. The 3CD expression cassette was inserted into plasmid YCA6-P1 at the Bgl II site by means of homologous recombination, resulting in plasmid YCA6-003.

1.3 Yeast transformation and selection

The plasmid YCA6-003 is linearized by a single enzyme cutting EcoNI site and is electrically transformed into the Pichia sink ^TM Strain 1 (Invitrogen). Both yeast transformation and subsequent selection of transformants were performed according to the product instructions. Transformed yeast clones were first subjected to small-scale culture and methanol induction. After induction, the bacterial solution was centrifuged to lyse the cells. The lysate supernatants were collected for ELISA and Western blotting analysis.

1.4 ELISA and immunoblotting experiments

And (3) carrying out enzyme-linked immunosorbent assay on the obtained lysate supernatant. Each well contained 5. Mu.l of lysate supernatant and 45. Mu.l of PBS solution coated 96-well Elisa plates overnight at 4 ℃, followed by incubation with 5% skim milk in PBST for 1h at 37 ℃, then with polyclonal antibodies against CA6VLP as primary antibody for 2h at 37 ℃, incubation with the corresponding horseradish peroxidase (HRP) -labeled secondary antibody for 1h, and reading the absorbance OD450 after termination of color development with TMB. Immunoblot assays detection assays were performed with specific polyclonal antibodies (anti-CA 6VP0, anti-CA 6VP1 and anti-CA 6VP 3) and their corresponding HRP-labeled secondary antibodies as previously described.

1.5 Expression and purification of VLPs

The expression and purification of CA6 VLPs was performed as reported in the literature (Zhang C, et al (2015) High-yield production of recombinant virus-like proteins of enterovirus 71in Pichia pastoris and the said protective infection in mice 33: 2335-2341). The empty vector pPink-HC was transformed into yeast as a control group, and the purified protein was used as a control antigen. The concentration of the finally obtained CA6 VLPs was determined by Bradford method.

1.6 Electron microscopy

After staining the CA6 virus-like particles with 0.5% uranyl acetate in water, it was further confirmed that the prepared samples had assembled to form complete virus-like particles by observation under a Tecnai G2 Spirit electron microscope.

1.7 mouse immunization and antibody detection

The purified CA6-VLP was diluted with 0.15M PBS, and then 6. Mu.g of CA6VLP or control antigen (300. Mu.l) was mixed well with an equal volume of aluminum adjuvant Alhydrogel (3 mg) (Invivogen, USA) and an equal volume of 1:1. Two groups (6 mice/group) of 6-week-old female ICR mice were intraperitoneally injected with 1. Mu.g of control antigen or CA6VLP, respectively. Immunizations were performed three times at 0,2,5. At 7,9 Zhou Caixie, sera were isolated for antibody detection and passive protection experiments in vivo. IgG antibodies specific for CA6 in serum were determined by ELISA experiments. Briefly, 96-well plates (10 ng/well) were coated with CA6 VLPs, or plates were coated with a mixture of CA6VP0, VP1, VP3 expressed in e.coli (1.

1.8 in vitro Cross-neutralization assay

Multiple tests prove that the CA6 virus cannot be amplified in conventional cell lines such as RD, vero, 293T, neuro-2A and the like for a long time, and sufficient high-titer virus cannot be obtained by in vitro culture, so that the in vitro neutralization effect of the CA6 virus is not evaluated by using the obtained antiserum. Only the neutralizing effect of serum on CA16, CA10 and EV71 viruses was examined, as follows: diluting the obtained immune serum at 16 ×,32 ×,64 ×,128 ×,256 ×,512 ×,1024 × and 2048 × times, adding 50 μ l/well into 96-well plate, adding 100TCID 50/well CA16, CA10 and EV71 virus, incubating at 37 deg.C for 1 hr, adding 10 ⁴ RD cells per well, the in vitro neutralizing effect of anti-CA 6-VLP sera against the three viruses was tested.

1.9 in vivo Passive protection experiment

Selecting four groups of ICR mice, injecting 50 μ l of anti-VLP serum or control group serum into the abdominal cavity, and injecting 1.469 × 10 into the abdominal cavity 24h later ⁶ Single copies of CA6/Gdula virus or 1.3383X 10 ⁴ One copy of the CA6/S0087b virus. Subsequently, the clinical status and survival of the mice were recorded for 15 consecutive days of observation. The clinical scoring criteria were as follows: 0, health; 1, slow movement; 2, ataxia; 3, paralysis; and 4, death. 2.9 statistical data statistical Difference analysis two-tailed t-tests were done using GraphPad Prism version 5 software.

Example 1 expression and purification of VLPs

In order to express CA6VLP, the present inventors inserted 3CD and P1 genes together into pPink-HC vector (FIG. 1A) to construct plasmid YCA6-003, then transformed Pichia pastoris competence with this plasmid, expressed in small amounts to obtain lysate centrifugation supernatant for ELISA and Western blotting analysis. The yeast clone transformed with the empty vector pPink-HC was run in parallel as a negative control. The yeast lysate supernatant transformed with plasmid YCA6-003 showed a clear antigen-antibody response compared to the control group (FIG. 1B). Three most strongly reactive yeast clones were selected for Western blotting analysis, and the experimental results are shown in the figure (fig. 1C), wherein anti-CA 6VP1, anti-CA 6VP0, and anti-CA 6VP3 were used as detection antibodies, the three yeast lysate samples all showed corresponding bands with sizes of 35kDa, 27kD, and 39kDa, respectively, and the control group showed no corresponding band. It has been demonstrated above that the yeast strain transformed with plasmid YCA6-003 is capable of expressing the P1 protein, and that 3CD is capable of cleaving P1 into VP0, VP1, VP3.

The YCA6-003 transformed yeast lysate was then subjected to sucrose density gradient centrifugation. Three bands, 39kDa, 35kD, and 27kDa in size, were clearly seen in layers #7, #8, and #9 (FIG. 2A). Western blotting analysis was performed on sucrose gradient samples, and three bands with sizes of 39kDa, 35kD and 27kDa were observed in layers #7, #8 and #9 using anti-CA 6VP1, anti-CA 6VP0 and anti-CA 6VP3 as detection antibodies, respectively (FIGS. 2B-D). The co-precipitation of VP0, VP1, VP3 at layers #7, #8 and #9 illustrates their co-assembly to form the VLP structure. Subsequently, when the layers #7, #8 and #9 were mixed together and observed under an electron microscope, a spherical particle structure having a diameter of about 30nm was observed (FIG. 2E). The above results indicate that pichia pastoris is capable of expressing the complete CA6 virus-like particle structure.

Example 2 mouse immunization

Before immunization of mice, CA6 VLPs and control antigens were prepared and quantified (fig. 3A), and subsequently mixed with aluminum adjuvant. Two groups (6/group) of ICR mice were intraperitoneally injected with CA6VLP vaccine and control antigen at 0,2,5, respectively. Blood was collected at week 7,9 for ELISA analysis. The plate was coated with the mixture of VP0, VP1 and VP3 at an equal ratio, and ELISA results are shown in FIG. 3B, in which 5 of 6 sera from the vaccine mice showed significant antigen-antibody reaction, while the control mice showed no significant reaction. Notably, when plates were packed with CA6 VLPs, all vaccine groups showed significant antigen-antibody responses (fig. 3C) and the geometric mean titer reached 17959 (fig. 3D). The above results not only demonstrate that CA6 VLPs are highly immunogenic, but also that the CA6 VLPs elicit antibodies that are directed primarily to conformational epitopes.

Example 3 in vitro Cross-neutralization assay of CA6-VLP sera

The obtained anti-CA 6 serum 1 was initially diluted 2-fold, and the results of cross-neutralization experiments on three viruses, CA16, CA10 and EV71, confirmed that the CA6 serum had no neutralizing cross-effect on the three viruses at the lowest dilution.

Example 4 in vivo passive protection assay of CA6VLP vaccine antiserum

The inventors evaluated the protective effect of anti-VLP antibodies by a serum passive protection assay. 7-day-old neonatal mice were challenged by intraperitoneal injection of 50 μ l of either anti-VLP serum or control serum and a lethal dose of CA6/Gdula or CA6/S0087B virus 24 hours later, with experimental results as shown in FIGS. 4A and 4B, and mice injected with control serum developed severe clinical symptoms including bradykinesia, ataxia and paralysis following challenge with CA6/Gdula virus and all died within 6d after challenge. In contrast, mice injected with anti-VLP serum did not show significant clinical symptoms during the 15 day observation period. Similarly, when challenged with CA6/S0087b virus, control mice all died within 9 days, and mice injected with anti-VLP serum all survived and exhibited no apparent clinical symptoms (FIGS. 4C-D). The above experimental results demonstrate that CA6 VLP-induced antibodies are able to confer complete in vivo protection in mice.

Discussion of the related Art

The P1 protein optimized codon shown in SEQ ID No.1 and the 3CD protein codon shown in SEQ ID No.9 can be efficiently expressed in pichia pastoris cells, and the expressed P1 protein can be cut by the 3CD protein to form capsid protein VP0 protein, VP1 protein and VP3 protein and form virus-like particles (VLP) in the cells. The purified virus-like particles (VLP) are used for immunizing mice, the immunogenicity is high, and the antibodies induced by the CA6VLP are mainly directed to conformational epitopes, the specificity is strong, and the mice can be protected from virus attack. Thus, the yeast-derived CA6VLP is a very potential candidate vaccine for CA6.

In recent years, the major pathogenic microorganisms causing HFMD are EV71, CA16 and CA6. The development of a multivalent vaccine with broad spectrum protective effect is now considered to be key to HFMD prevention. Recently, EV71 VLPs and CA16 VLPs were successfully expressed in pichia pastoris and animal experiments have validated their protective effects. Therefore, pichia pastoris expressing CA6 VLPs, EV71 VLPs and CA16 VLPs can form a trivalent vaccine, which would have a very positive impact on prevention of HMFD.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims (7)

1. An expression vector comprising a polynucleotide encoding a coxsackie A6 virus P1 protein; and the sequence of the polynucleotide is shown as SEQ ID NO.3, the expression vector also comprises a polynucleotide sequence for coding the coxsackie A6 virus 3CD protein, the expression vector comprises a first expression cassette and a second expression cassette, and the first expression cassette comprises the polynucleotide shown as SEQ ID NO.3 or the complementary sequence thereof; the second expression cassette comprises the polynucleotide shown in SEQ ID NO.8 or a complementary sequence thereof, the first expression cassette also comprises a promoter, the promoter is positioned at the upstream of the polynucleotide shown in SEQ ID NO.3, and the promoter is a PAOX1 promoter; and/or the second expression cassette further comprises a promoter, the promoter is positioned at the upstream of the polynucleotide shown in SEQ ID NO.8, and the promoter is PAOX1 promoter.

2. The expression vector of claim 1, wherein the expression vector is a recombinant baculovirus.

3. A host cell comprising the expression vector of claim 1, wherein the host cell is a yeast cell.

4. A method of preparing a coxsackievirus A6VLP comprising the steps of:

culturing the cell of claim 3 under conditions suitable for expression, thereby expressing a virus-like particle (VLP); and isolating the virus-like particle (VLP) expressed by the host cell of claim 3.

5. The method of claim 4, wherein the virus-like particle comprises polypeptides set forth in SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 6.

6. The method of claim 4, wherein said virus-like particle consists of the polypeptides set forth in SEQ ID No.4, SEQ ID No.5, and SEQ ID No. 6.

7. A pharmaceutical composition comprising a Virus Like Particle (VLP) or an expression vector of claim 1 or a host cell of claim 3, and a pharmaceutically acceptable carrier and/or adjuvant;

the virus-like particle is expressed by the host cell of claim 3.

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