阅读说明：本技术 密码子优化的小反刍兽疫病毒f基因核酸疫苗 (Codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine ) 是由 夏俊 汪萍 苗书魁 杜玮 马文戈 黄炯 金映红 沙吾尔江·阿不都力艾拉 赵洁雅 王 于 2019-10-29 设计创作，主要内容包括：本发明涉及分子生物学技术领域,具体的涉及一种密码子优化的小反刍兽疫病毒F基因核酸疫苗。该密码子优化基因序列兼顾了哺乳动物细胞密码子使用偏好,序列如SEQ ID NO.2所示。所涉及的小反刍兽疫病毒F基因核酸疫苗,在剔除F基因5′端信号肽后,将剩余序列经密码子优化,在5′端连接乙型脑炎病毒信号肽序列,最后将该序列插入真核表达载体中。该核酸疫苗在免疫小鼠后有效地刺激了免疫系统,产生了中和抗体,体现了良好的免疫原性。(The invention relates to the technical field of molecular biology, in particular to a codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine. The codon optimized gene sequence gives consideration to the codon usage preference of mammalian cells, and the sequence is shown as SEQ ID NO. 2. The related peste des petits ruminants virus F gene nucleic acid vaccine is characterized in that after a signal peptide at the 5 'end of an F gene is removed, the rest sequence is optimized through a codon, the 5' end is connected with a encephalitis B virus signal peptide sequence, and finally the sequence is inserted into a eukaryotic expression vector. The nucleic acid vaccine effectively stimulates an immune system after immunizing a mouse, generates a neutralizing antibody and embodies good immunogenicity.)

1. A codon optimized Peste des petits ruminants virus F gene sequence is shown in SEQ ID NO. 2.

2. Use of the codon-optimized Peste des petits ruminants virus F gene sequence of claim 1 in the preparation of a vaccine for the prevention of Peste des petits ruminants.

3. A codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine is obtained by removing a signal peptide from a PPRVF gene sequence shown in SEQ ID No.2, optimizing the rest sequence by a codon and inserting the rest sequence into a eukaryotic expression vector.

4. The nucleic acid vaccine of claim 3, wherein: the eukaryotic expression vector is pcDNA3.1 (+).

5. The nucleic acid vaccine of claim 4, wherein: the PPRV F gene nucleic acid vaccine is obtained by inserting a PPRV F gene sequence shown by SEQ ID NO.2 between BamH I and EcoR I enzyme cutting sites of a eukaryotic expression vector pcDNA3.1(+) after removing a signal peptide and optimizing a codon of a residual sequence; the 5' end is connected with a signal peptide sequence of the encephalitis B virus.

Technical Field

The invention relates to the technical field of molecular biology, in particular to a codon-optimized peste des petits ruminants virus F gene nucleic acid vaccine.

Background

Peste-des-peptides (PPR) is a serious virulent, contact infectious disease caused by Peste-des-peptides virus (PPRV) and is characterized by fever, erosion of oral and tongue mucous membranes, lacrimation, rhinorrhea, diarrhea and pneumonia. PPR is a group a virulent infectious disease that must be reported by the world animal health Organization (OIE) regulations. Sheep and goats are susceptible to infection, and cattle, buffalo, pigs and wild animals can also be infected. PPRV infection can lead to sheep and goats with up to 100% morbidity and significant mortality, severely impacting food safety and herdsman survival, especially in developing countries that rely on small ruminants. Since the first outbreak of PPR in 1942 was in kortewa, it was prevalent mainly in east africa, west africa, central africa, arabian peninsula, south asia, and so on. In 7 months in 2007, PPR is firstly discovered in the region of Tibetan Ali in China, and until 9 months in 2014, PPR epidemic situations occur in 256 counties of 22 provinces (autonomous regions) in China, so that economic losses are large and social influences are serious. The global program is to eliminate Peste des petits ruminants by 2030. The Ministry of agriculture in China publishes' the national Peste des petits ruminants elimination plan (2016 + 2020), which aims at meeting the standard of non-immune and non-epidemic areas of Peste des petits ruminants nationwide by the plan of 2020. Therefore, it is important to strengthen the basic research on the application aspect of PPRV.

PPRV belongs to the genus morbillivirus of the family Paramyxoviridae, is a single-stranded negative-strand nonsegmented RNA virus with a genome total length of 15948bp and consists of 6 structural proteins (C and V) and 2 nonstructural proteins (C and V) of a nucleocapsid protein (N), a phosphoprotein (P), a coding matrix protein (M), a fusion protein (F), a hemagglutinin protein (H) and a large protein (L). The F protein is related to the adsorption and invasion of the virus, is one of the key factors for determining whether the virus can successfully infect a host, and is a main protective immunogen for inducing the formation of neutralizing antibodies. The function of the F protein is to assist the virus in entering the host cell. Once the virus binds to a target in the host cell, the F protein regulates fusion of the viral membrane and the cell membrane and allows the directly released N protein to enter the cytosol. During viral infection, newly synthesized fusion protein molecules fuse cells to each other, and because of this mechanism, inhibition of the activity of the F protein is important to prevent paramyxovirus infection.

Nucleic acid vaccine (nucleic vaccine), gene vaccine (gene vaccine) or DNA vaccine (DNAvcine) refers to cloning exogenous gene into eukaryotic expression vector, and introducing recombinant plasmid into animal body to make exogenous gene synthesize antigen protein via transcription system of host cell, activate immune system of body and initiate immune reaction. In 1996, the world health organization uniformly named them as nucleic acid vaccines. It has the following advantages: can fully simulate the natural infection state, synthesize the protein with corresponding space conformation in the eukaryotic cell, not only stimulate the organism to generate humoral immune response, but also induce the immune response generating specific CD8+ cytotoxic lymphocyte (CTL); compared with recombinant virus live vector vaccines which have many self proteins to be expressed besides expression target genes, nucleic acid vaccines only express antigen genes on the vectors in cells, and the vectors have no immunogenicity, do not stimulate organisms to generate antibodies, and can better ensure the purity of the antigen proteins; the nucleic acid vaccine can avoid directly contacting pathogens with higher risk, and can optimize and modify codons at the DNA level, so that the nucleic acid vaccine can be more efficiently expressed and more effectively stimulate the immune system of an organism; and the nucleic acid vaccine has low cost and good stability, and is suitable for large-scale production, transportation, storage and the like. However, the nucleic acid vaccine has many disadvantages, the most important of which is that the object of research and application of the nucleic acid vaccine is mainly eukaryote, and the most of the target genes come from prokaryotes such as virus or bacteria, and the prokaryotes and eukaryotes have obvious differences in codon usage preference, which results in that the foreign genes of the nucleic acid vaccine cannot be efficiently expressed in eukaryotic cells and cannot effectively stimulate the immune system of the body to generate response.

Disclosure of Invention

The invention aims to overcome the defects and provide a codon-optimized PPRV F gene sequence.

Another objective of the invention is to provide a PPRV F gene nucleic acid vaccine carrying Japanese Encephalitis Virus Signal Peptide (JEVSP) on an optimized basis.

The purpose of the invention is realized by the following technical scheme:

a PPRVF gene sequence with optimized codon, which is SEQ ID NO. 2. The original PPRVF gene sequence is SEQ ID NO. 1.

The application of the codon-optimized PPRV F gene sequence shown in SEQ ID NO.2 in the preparation of Peste des petits ruminants vaccines.

A PPRV F gene nucleic acid vaccine is obtained by removing signal peptide from PPRV F gene sequence shown in SEQ ID NO.2, optimizing the rest sequence by codon, and inserting into eukaryotic expression vector.

Wherein, the eukaryotic expression vector is pcDNA3.1 (+); the PPRV F gene nucleic acid vaccine is obtained by firstly removing a signal peptide from a PPRV F gene sequence shown by SEQID NO.2, optimizing a remaining sequence by a codon, and inserting the rest sequence between BamH I and EcoR I enzyme cutting sites of a eukaryotic expression vector pcDNA3.1 (+); the 5' end is connected with a signal peptide sequence of the encephalitis B virus.

The construction steps of the codon-optimized PPRV F gene sequence and the nucleic acid vaccine thereof provided by the invention are as follows:

1. construction of original sequence PPRV F gene sequence nucleic acid vaccine

Using the PPRV Nigeria/75/1 strain as a reference sequence (GenBank: X74443), the F gene with a total length of 1641bp was selected, synthesized by Biotechnology engineering (Shanghai) GmbH, and loaded into the vector pUC57 to form pUC 57-F. pUC57-F was digested with BamH I and XbaI, and the target fragment was recovered and purified with a DNA gel recovery kit (AxyPrep DNA gel recovery kit, Cat. No.: AP-GX-250) for use in the construction of a nucleic acid vaccine. The sequence of the PPRV F gene before codon optimization is SEQ ID NO. 1.

2. Construction of codon-optimized PPRV F gene sequence nucleic acid vaccine without signal peptide

(1) Design and Synthesis of codon-optimized Signal peptide-free PPRV F Gene sequences

PPRV Nigeria/75/1 strain is taken as a reference sequence (GenBa)nk: X74443), selecting F gene with the total length of 1641bp, removing the residual 1584bp after the signal peptide (57bp) at the 5' end is removed, and then applying software OptimumGene^TMAnalyzing the gene sequence, finding out the codon usage preference of the gene sequence, simultaneously finding out the codon sites with the codon usage preference different from that of sheep, replacing the codons with the different usage preferences in the PPRV F gene by the codons favored by sheep, and then designing the PPRV F gene sequence with optimized codons. The gene is chemically synthesized by the company of Biotechnology engineering (Shanghai), and the amino acid sequence of the protein coded by the PPRV F gene sequence which rejects the signal peptide and is codon-optimized is consistent with the amino acid sequence of the F gene which rejects the signal peptide and is not codon-optimized. The F gene sequence after codon optimization is SEQ ID NO. 2.

(2) A recombinant vector pUC57-F-opt containing a target sequence provided by the company Limited in the biological engineering (Shanghai) is subjected to double enzyme digestion by BamH I and EcoR I, and a DNA gel recovery kit (AxyPrep DNA gel recovery kit, Cat. No.: AP-GX-250) is used for recovering and purifying a target fragment, wherein the target fragment is a PPRVF gene sequence with optimized codons after F gene signal peptides are removed, and two ends of the target fragment are respectively connected with gene sequences of BamH I enzyme digestion sites and EcoR I enzyme digestion sites so as to facilitate the construction of a nucleic acid vaccine.

3. Construction of codon-optimized PPRV F gene sequence nucleic acid vaccine containing signal peptide

(1) Design and synthesis of codon-optimized PPRV F gene sequence containing signal peptide

The PPRV F gene sequence which is designed in the 2(1) and optimized by the codon is connected with a Japanese Encephalitis Virus Signal Peptide (JEVSP) sequence (the total length is 72bp) at the 5' end. The gene is obtained by chemical synthesis from the company of Biotechnology engineering (Shanghai). The sequence of the Japanese Encephalitis Virus Signal Peptide (JEVSP) is SEQ ID NO. 3.

(2) A recombinant vector pUC57-JEVSP-F containing a target sequence provided by the company Limited in the engineering bioengineering (Shanghai) is subjected to double enzyme digestion by BamH I and EcoR I, and a DNA gel recovery kit (AxyPrep DNA gel recovery kit, Cat. No.: AP-GX-250) is used for recovering and purifying a target fragment, wherein the fragment is a PPRV F gene sequence optimized by codons, and both ends of the fragment are respectively connected with BamH I enzyme digestion sites and EcoR I enzyme digestion sites so as to facilitate the construction of a nucleic acid vaccine.

4. Cloning the gene fragment obtained in the step 1 into a eukaryotic expression vector pcDNA3.1(+) to obtain a recombinant plasmid pcDNA3.1(+) -F. Cloning the gene fragment obtained in the step 2 into a eukaryotic expression vector pcDNA3.1(+) to obtain a recombinant plasmid pcDNA3.1(+) -F-opt. Cloning the gene fragment obtained in the step 3 into a eukaryotic expression vector pcDNA3.1(+) to obtain a recombinant plasmid pcDNA3.1(+) -JEVSP-F-opt. After extracting, enzyme cutting and sequencing the recombinant plasmid, determining to obtain a plasmid which is consistent with expectation, namely the PPRV F gene nucleic acid vaccine.

Has the advantages that: compared with a wild gene, the codon-optimized PPRVF gene sequence has increased codon occurrence frequency preferred by sheep cells, but the amino acid sequence of the encoded PPRVF protein is unchanged, so that the PPRVF gene sequence is more suitable for protein expression in the sheep cells and is more beneficial to stimulating the body to generate immune response.

Since there is no unified standard or principle for codon optimization of gene sequences, different researchers will design different nucleotide sequences for expression of target proteins, and accordingly there may be significant differences in expression efficiency. According to the optimized nucleotide coding sequence, the defects are overcome, and the PPRV F protein expression level is improved; the optimized gene is used for constructing a nucleic acid vaccine, so that an immune system is more effectively stimulated after a mouse is immunized, a neutralizing antibody is generated, and good immunogenicity is reflected. Meanwhile, on the basis of codon optimization, the sequence is connected with a (JEVSP) signal peptide, so that the secretion of the protein is promoted more effectively, and the capacity of inducing the antibody generation by the target protein is improved.

The PPRV F gene optimized by codon and a (JEVSP) signal peptide sequence are directly cloned into a eukaryotic expression vector, and PPRV nucleic acid vaccines pcDNA3.1(+) -F-opt and pcDNA3.1(+) -JEVSP-F-opt are constructed, and animals immunized by the two vaccines can be stimulated to generate neutralizing antibodies. And compared with the pcDNA3.1(+) -JEVSP-F-opt, the pcDNA3.1(+) -JEVSP-F-opt introduced with the JEVSP signal peptide can more effectively generate neutralizing antibody. Therefore, the pcDNA3.1(+) -F-opt and the pcDNA3.1(+) -JEVSP-F-opt nucleic acid vaccines have good immunogenicity.

Drawings

FIG. 1 shows the restriction enzyme electrophoresis pattern of recombinant plasmids pcDNA3.1(+) -F, pcDNA3.1(+) -F-opt, and pcDNA3.1(+) -JEVSP-F-opt; among them, lane M, GeneRuler 1kb Plus DNA Ladder; lane B, plasmid pcDNA3.1(+) -F by BamH I and Xba I double enzyme electrophoresis map; lane C, plasmid pcDNA3.1(+) -F-opt by BamH I and EcoR I double enzyme electrophoresis map; lane D, double-restriction electrophoretogram of plasmid pcDNA3.1(+) -JEVSP-F-opt by BamH I and EcoR I.

Detailed Description

All reagents, materials and equipment used in the present invention are well known in the art and are not intended to limit the practice of the present invention, and other reagents and equipment well known in the art may be used in the practice of the following embodiments of the present invention.