CN108329386A - A kind of antiviral signal protein of goose source mitochondria and its application - Google Patents

Abstract

The present invention relates to a kind of antiviral signal protein of goose source mitochondria and its applications, include the combination of any one or at least two in active region, proline-rich region, structural domain A, structural domain B or transmembrane region more particularly to a kind of antiviral signal protein of goose source mitochondria with antiviral and immune activation function and its application, the signal protein.The antiviral signal protein of goose source mitochondria the innate antiviral of induced high levels can react in primary goose embryo fibroblast, and the early stage of newcastle disease virus can be inhibited to replicate.

Description

A kind of antiviral signal protein of goose source mitochondria and its application

Technical field

The present invention relates to gene and protein engineering field more particularly to a kind of antiviral signal protein of goose source mitochondria and It is applied, and in particular to a kind of to have antiviral and immune activation function the antiviral signal protein of goose source mitochondria and its answer With.

Background technology

By the cause of disease model molecule of pattern recognition receptors (PRR) identification virus, induction generates interferon (IFN) and inflammatory Factor suppressing virus replication is the most important a kind of innate antiviral mechanism of mammal.And know in all conservative patterns In other receptor, Induced by Retinoic Acid gene protein I (RIG-I) and Differentiation of Human Melanoma Cell Line related gene 5 (MDA5) are most important special Industry identifies the cytoplasm pattern recognition receptors of viral RNA.RIG-I and MDA5 is similar two kinds of receptors, and identification is different types of Viral RNA, they have the identical antiviral signal protein of downstream signal linkers mitochondria (MAVS), MAVS to feel in virus The congenital immunity for contaminating induction plays pivotal role in the process.

" adjustment effect of the antiviral signal protein of mitochondria (MAVS) in host's innate immunity signal path, Yang Xingxing Deng, Progress in Biochemistry and Biophysics, 2013,40 (5):Toll-like receptor (TLR) and RIG-I are reported in 397-405 " (RLR) etc. the pathogen of cell patterns identification receptor identification invasion and signal is passed into MAVS, MAVS, which passes through, stimulates downstream TBK1 complexs and IKK complexs activate the signal paths such as NF- κ B and IRF3 respectively, and then activate the expression of interferon α/β, induce Intracellular anti-infective innate immune reaction.But it is not specifically described influences of the MAVS of separate sources for different animals, Compared with mammal, the immunogene total amount of birds is less, it is meant that birds congenital immunity has the uniqueness of its own, no Only in this way, the innate immune mechanisms between birds are also not quite similar, such as chicken lacks important RNA pattern recognition receptors RIG- I, but duck and goose then has RIG-I, and congenital immunity molecular function has larger difference between illustrating different species.

ND (newcastle disease, Newcastle disease) is a kind of birds epidemic disease of acute height lethal, is a kind of to fowl Class has the infectious disease of highly infectious and lethal, since chicken is most susceptible birds, so the disease often aviculture is caused it is huge Big loss.The cause of disease of ND is newcastle disease virus (New castle disease virus, NDV), is paramyxovirus section (Paramyxoviridae), fowl Rubulavirus (Avulavirus) member.NDV is sub-thread minus-stranded rna virus, genome Overall length about 15.8kb, including 6 open reading frames are separately encoded nucleoprotein (NP), phosphoprotein (P), stromatin (M), melt Hop protein (F), hemagglutinin-neuraminidase (HN) and big polymerase protein (L).According to Virulence Difference, NDV can be divided into strong poison Strain (Velogenic), mesogenic strains (Mesogenic) and low virulent strain (Lentogenic).Velogen strain can cause fowl it is acute, Lethal infection has the higher death rate.Low virulent strain only causes slight respiratory tract infection or enteric infection, thus extensive Ground applies to the production of live Newcastle disease vaccine.

Goose China as a kind of important economic animal, have raising throughout our country, although goose as aquatic bird in recent years Constantly have the report that the virosis such as ND occur, but relative to terrestrial birds, has stronger resistance to newcastle disease virus (NDV) Power.MAVS is as most important hinge molecule in RIG-I signal paths, effect during goose is antiviral and immune activation It is unclear.

Invention content

The purpose of the present invention is to provide a kind of antiviral signal protein of goose source mitochondria and its application, the goose source mitochondrias Antiviral signal protein the innate antiviral of induced high levels can react in primary goose embryo fibroblast, and can press down The early stage of newcastle disease virus processed replicates.

To reach the invention purpose, the present invention uses following technical scheme：

In a first aspect, the present invention provides the antiviral signal protein of a kind of goose source mitochondria (MAVS), the signal protein Including any one in active region (CARD), proline-rich region (areas PRR), structural domain A, structural domain B or transmembrane region (TM) Kind or at least two combination.

In the present invention, inventor amplifies goose source MAVS, find goose source MAVS include active region, proline-rich region, In structural domain A, structural domain B or transmembrane region any one or at least two combination, have special antivirus action, can It plays a significant role in congenital immunologic process.

According to the present invention, for the amino acid sequence of the active region as shown in SEQ ID NO.1, particular sequence is as follows：

GHILRNMDKFKNIHVASLVDSLSCLTEADRDELHTRGEVRGSNATAYKLYQHVKCRRGWVTDLINALH；

According to the present invention, the amino acid sequence of the proline-rich region is as shown in SEQ ID NO.2, and particular sequence is such as Under：

SAPPAAPNPRVASFPPDAQPVSAQLPFSGPKPAADAPLADPPRCTSPTGGHPPLQRAAATAASPAGSSAASVDVPST DLDARAPVQEKPLEQKSPQPPLQPLSAARAGASDGQS；

According to the present invention, for the amino acid sequence of the structural domain A as shown in SEQ ID NO.3, particular sequence is as follows：

AAGVVLPPAAPPEQGREWLGQRYPVCVDNGCFGNAKHLRRAAPGSGLGTAVPLRD；

According to the present invention, for the amino acid sequence of the structural domain B as shown in SEQ ID NO.4, particular sequence is as follows：

SVGAAGRPTSPPGEFCHKHLGLSHHPGGGRGAQQAGRPPLRGGRQPAGSRPAPGRLRFRGHRSPGLEERPAPAEHRQ LRSRRSTRGIRLGTLLLGILRRRSGGSTLLVPPELENRRDPRGSRPQRPAEGEQRRPGRRSRSLGRPPGFRLERRQR RHAWQRPQPWGQPRAVPALPRPGRGHRAHLRRGVPGVRPAAE；

According to the present invention, for the amino acid sequence of the transmembrane region as shown in SEQ ID NO.5, particular sequence is as follows：

AAAAPREVPLPGRNPVWRV；

According to the present invention, the signal protein include active region, proline-rich region, structural domain A, structural domain B and across Film area, the amino acid sequence of the signal protein as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.7, Specific sequence is as follows：

The amino acid sequence (SEQ ID NO.6) of MAVS： MGFAEDKVYGHILRNMDKFKNIHVASLVDSLSCLTEADRDELHTRGEVRGSNATAYKLYQHVKCRRGWVTDLINALH QNNAGHLAEELQQVYDLYQTPPPTASAPPAAPNPRVASFPPDAQPVSAQLPFSGPKPAADAPLADPPRCTSPTGGHP PLQRAAATAASPAGSSAASVDVPSTDLDARAPVQEKPLEQKSPQPPLQPLSAARAGASDGQSARGPLPCPSEAQQAA AGPPGAAGVVLPPAAPPEQGREWLGQRYPVCVDNGCFGNAKHLRRAAPGSGLGTAVPLRDPVTARGAGQPRNEPQED VYVSTELPAGLAEPLCGEGVPQPRGPQGTQEKQAELPEHRGPPSSFVDVRSPLLIQQQFDAEQKRVGTLREHEEGAT RMETIPPAAAPRDTSPSRDVSWKLPVPEEALPAGKAAGSTPSVPAEEKVLPASAAPLQGPAVGGSSVGAAGRPTSPP GEFCHKHLGLSHHPGGGRGAQQAGRPPLRGGRQPAGSRPAPGRLRFRGHRSPGLEERPAPAEHRQLRSRRSTRGIRL GTLLLGILRRRSGGSTLLVPPELENRRDPRGSRPQRPAEGEQRRPGRRSRSLGRPPGFRLERRQRRHAWQRPQPWGQ PRAVPALPRPGRGHRAHLRRGVPGVRPAAEIAESNGGAAAAPREVPLPGRNPVWRV；

The nucleotide sequence (SEQ ID NO.7) of MAVS： atgggctttgcggaggacaaggtgtacggccacatcctgaggaacatggacaagttcaagaacatccacgtggcgtc gctggtcgattccctcagctgcctcaccgaagccgacagggacgaacttcatacccgaggggaggtacggggcagca atgcaaccgcctacaagttatatcagcacgtgaagtgccgccggggctgggtgaccgacctgatcaacgcgctgcac cagaacaacgcggggcacctggccgaagagctgcagcaggtctatgacctctaccaaacccctcctccaacagcctc agctccaccagctgctccaaatcctcgtgtggcctccttccctcctgatgcccagcccgtcagtgcccagctgccat tctcgggccccaagcccgctgcggacgccccgctggccgatcctccccgctgcacctcgcccaccggcggccatcca cctctccagcgcgctgctgccaccgctgcgagcccagccgggagctcagctgcgagcgtggacgttcccagcaccga cctggacgccagggcccctgtgcaggagaagcccctggaacaaaagagtccccagccaccgctgcagccgctgagcg cggcccgtgctggagcgagcgacgggcagagcgcgcgggggccgctcccgtgcccctccgaggctcagcaggcggca gcggggcccccgggggctgccggcgtggttttgccgccggctgctccccccgagcagggccgggagtggctgggcca gcgctacccggtgtgcgtggacaacgggtgcttcgggaacgccaagcacctgcgccgcgccgcgccgggctcggggc tgggcacggccgtcccgctgagggacccggtcaccgctcgtggcgccgggcagcccaggaacgagccccaggaagac gtctacgtctccaccgagttgcccgcggggttggcagagcctctgtgcggcgagggggtgccgcagccccgcggccc gcaggggacgcaggaaaagcaggcggagctccccgagcaccgagggcctccgagcagcttcgtggacgtgcgcagcc ccctcctcatacagcagcagtttgacgcggagcagaagcgggtcgggacgctgcgagagcacgaagagggagccacc cggatggaaacaatccccccggccgccgcccccagagacacttccccatcccgcgacgtctcctggaagcttcccgt gccagaggaagcgctgcctgcggggaaggcggccggcagcaccccctccgtgccggcagaggagaaagtgctcccgg cctcggcggcccctctccagggcccagcggtcgggggaagctctgtgggcgcagctgggaggccgacgtcccccccg ggggagttctgccacaagcatctgggcctctcccaccatcccggaggaggacgcggagctcagcaagccgggcgtcc tcctctccgtggcgggcgacagcccgcaggcagccgcccggcccccgggcggctccgcttccgaggccaccgatcac ctgggcttgaggagcgacccgctcctgctgagcaccgacagctcaggtcccggaggagcacgcgagggatccgcctc gggacgttgctcctcggcatcctgcggagacgaagcggggggagcacgctcctggtcccccccgagctggagaaccg acgagatccgcgtggctcacggccccagcgccctgctgaaggcgagcaacgacgtcccggacgacggagccggtccc tcgggcggccccccggcttccgcctcgagcggaggcagcgacgccacgcctggcagcgtccccagccctggggacag ccccgggccgtccctgccctacctcgtcccggccgtgggcatcgcgctcatctccgccgtggcgttcctggtgtacg cccggctgcagaaatagcggagagcaacgggggtgccgcggccgccccgcgggaggttcctcttcccggcaggaatc ccgtctggcgcgtttga.

In the present invention, the mRNA sequence of goose source MAVS genes is expanded, overall length 2019bp has found same with NCBI forecasting sequences Source property is up to 96.2%, but wherein has the insertion of three sections of sequences, i.e., active region (areas CARD), TM (transmembrane region) and proline are rich Ji Qu (areas PRR), length function goose source MAVS and play an important role up to 828 bases.

According to the present invention, the signal protein includes active region, structural domain A, structural domain B and transmembrane region, the signal The amino acid sequence of albumen is as shown in SEQ ID NO.8, and nucleotide sequence is as shown in SEQ ID NO.9, and specific sequence is such as Under：

Lack the amino acid sequence (SEQ ID NO.8) of the MAVS in the areas PRR： MGFAEDKVYGHILRNMDKFKNIHVASLVDSLSCLTEADRDELHTRGEVRGSNATAYKLYQHVKCRRGWVTDLINALH QNNAGHLAEELQQVYDLYQTPPPTAARGPLPCPSEAQQAAAGPPGAAGVVLPPAAPPEQGREWLGQRYPVCVDNGCF GNAKHLRRAAPGSGLGTAVPLRDPVTARGAGQPRNEPQEDVYVSTELPAGLAEPLCGEGVPQPRGPQGTQEKQAELP EHRGPPSSFVDVRSPLLIQQQFDAEQKRVGTLREHEEGATRMETIPPAAAPRDTSPSRDVSWKLPVPEEALPAGKAA GSTPSVPAEEKVLPASAAPLQGPAVGGSSVGAAGRPTSPPGEFCHKHLGLSHHPGGGRGAQQAGRPPLRGGRQPAGS RPAPGRLRFRGHRSPGLEERPAPAEHRQLRSRRSTRGIRLGTLLLGILRRRSGGSTLLVPPELENRRDPRGSRPQRP AEGEQRRPGRRSRSLGRPPGFRLERRQRRHAWQRPQPWGQPRAVPALPRPGRGHRAHLRRGVPGVRPAAEIAESNGG AAAAPREVPLPGRNPVWRV；

Lack the nucleotide sequence (SEQ ID NO.9) of the MAVS in the areas PRR： atgggctttgcggaggacaaggtgtacggccacatcctgaggaacatggacaagttcaagaacatccacgtggcgtc gctggtcgattccctcagctgcctcaccgaagccgacagggacgaacttcatacccgaggggaggtacggggcagca atgcaaccgcctacaagttatatcagcacgtgaagtgccgccggggctgggtgaccgacctgatcaacgcgctgcac cagaacaacgcggggcacctggccgaagagctgcagcaggtctatgacctctaccaaacccctcctccaacagccgc gcgggggccgctcccgtgcccctccgaggctcagcaggcggcagcggggcccccgggggctgccggcgtggttttgc cgccggctgctccccccgagcagggccgggagtggctgggccagcgctacccggtgtgcgtggacaacgggtgcttc gggaacgccaagcacctgcgccgcgccgcgccgggctcggggctgggcacggccgtcccgctgagggacccggtcac cgctcgtggcgccgggcagcccaggaacgagccccaggaagacgtctacgtctccaccgagttgcccgcggggttgg cagagcctctgtgcggcgagggggtgccgcagccccgcggcccgcaggggacgcaggaaaagcaggcggagctcccc gagcaccgagggcctccgagcagcttcgtggacgtgcgcagccccctcctcatacagcagcagtttgacgcggagca gaagcgggtcgggacgctgcgagagcacgaagagggagccacccggatggaaacaatccccccggccgccgccccca gagacacttccccatcccgcgacgtctcctggaagcttcccgtgccagaggaagcgctgcctgcggggaaggcggcc ggcagcaccccctccgtgccggcagaggagaaagtgctcccggcctcggcggcccctctccagggcccagcggtcgg gggaagctctgtgggcgcagctgggaggccgacgtcccccccgggggagttctgccacaagcatctgggcctctccc accatcccggaggaggacgcggagctcagcaagccgggcgtcctcctctccgtggcgggcgacagcccgcaggcagc cgcccggcccccgggcggctccgcttccgaggccaccgatcacctgggcttgaggagcgacccgctcctgctgagca ccgacagctcaggtcccggaggagcacgcgagggatccgcctcgggacgttgctcctcggcatcctgcggagacgaa gcggggggagcacgctcctggtcccccccgagctggagaaccgacgagatccgcgtggctcacggccccagcgccct gctgaaggcgagcaacgacgtcccggacgacggagccggtccctcgggcggccccccggcttccgcctcgagcggag gcagcgacgccacgcctggcagcgtccccagccctggggacagccccgggccgtccctgccctacctcgtcccggcc gtgggcatcgcgctcatctccgccgtggcgttcctggtgtacgcccggctgcagaaatagcggagagcaacgggggt gccgcggccgccccgcgggaggttcctcttcccggcaggaatcccgtctggcgcgtttga.

In the present invention, inventor has found that the scarce goose MAVS in the areas missing PRR can induce higher levels of interferon stimulation base The expression of cause illustrates that the areas PRR play the effect of negative regulation MAVS functions, and the goose MAVS for lacking PRR can be used as one kind preferably Immunostimulant.

Second aspect, the present invention provide a kind of preparation of antiviral signal protein of goose source mitochondria as described in relation to the first aspect Method includes the following steps：Using the genome of existing goose on GenBank and the MAVS sequences of goose MAVS predictions as template, Amplimer is designed, is expanded using the method for over-lap PCR.

Preferably, for the nucleotide sequence of the primer as shown in SEQ ID NO.10-13, particular sequence is as follows：

P1(SEQ ID NO.10)：ATGGGCTTTGCGGAGGACAAG；

P2(SEQ ID NO.11)：CTGGCACGGGAAGCTTCCAGGAGACGTCGC；

P3(SEQ ID NO.12)：GCGACGTCTCCTGGAAGCTTCCCGTGCCAG；

P4(SEQ ID NO.13)：TCAAACGCGCCAGACGGGATTCCT；

In the present invention, P1, P2 are the upstream and downstream primer of first round PCR, expand MAVS genes front 1200bp or so segment, P3, P4 are the upstream and downstream primer of the second wheel PCR, the segment of amplification MAVS genes rear portion 1800bp or so, the mixing of two-wheeled PCR product Third round PCR is carried out using P1 and P4 obtain full length sequence, connection carrier T sequencing afterwards.

The third aspect, the present invention provide the gene for encoding the antiviral signal protein of goose source mitochondria as described in relation to the first aspect.

Fourth aspect, the present invention provide a kind of recombinant plasmid, and it includes the genes described in the third aspect.

5th aspect, the present invention provide a kind of recombinant virus, and it includes the genes described in the third aspect.

6th aspect, the present invention provide a kind of vaccine adjuvant, and it includes the recombinant plasmids or the such as the 5th described in fourth aspect Recombinant virus described in aspect.

In the present invention, MAVS is inserted into NDV genomes using reverse Genetics Technique and builds MAVS recombinant viruses, or It is used as vaccine adjuvant using 5 ' ppp RNA of access differential stimulus object 21-mer or by protokaryon great expression MAVS, is used for Interferon is generated using initial stage induction in vaccine, supports viral infection resisting.

7th aspect, the present invention provide a kind of host cell, it includes the recombinant plasmid described in fourth aspect, the host Cell is Escherichia coli.

Eighth aspect, the present invention provide the gene as described in the third aspect, the recombinant plasmid as described in fourth aspect, such as the Recombinant virus described in five aspects or the vaccine adjuvant as described in terms of the 6th is used to prepare antiviral and/or Immune-enhancing effect medicine Object.

According to the present invention, the virus is newcastle disease virus.

In the present invention, inventor has found that goose source MAVS can induce the elder generation of highest level in primary goose embryo fibroblast Nature antiviral response, and the early stage of newcastle disease virus can be inhibited to replicate.

Compared with prior art, the invention has the advantages that：

(1) present invention is by expanding goose source MAVS, it is determined that goose source MAVS genes, overall length 2019bp have found pre- with NCBI It surveys sequence homology and is up to 96.2%, it is potential that verification finds that the goose source MAVS and goose source MAVS for lacking PRR structural domains can be used as Goose immunostimulant and antiviral gene；

(2) goose source MAVS can induce the innate antiviral of highest level in primary goose embryo fibroblast in the present invention Poison reaction, and the early stage of newcastle disease virus can be inhibited to replicate.

Description of the drawings

Fig. 1 is E Yuan, Ji Yuan of the present invention, Ya Yuan, people source and the MAVS sequence alignment results in mouse source；

Fig. 2 goose source MAVS of the present invention and NCBI prediction geese source MAVS sequence alignment results；

Fig. 3 is that the present invention is based on the phylogenetic analysis of each species MAVS sequences；

Fig. 4 is present invention structure each structural domain deletion plasmid ideographs of goose source MAVS；

Fig. 5 (A) is the table of E Yuan, Ji Yuan and people source MAVS in goose fibroblast inducing interferon stimulated gene IFITM3 Up to level, Fig. 5 (B) is the expression water of E Yuan, Ji Yuan and people source MAVS in goose fibroblast inducing interferon stimulated gene MX Flat, Fig. 5 (C) is the expression of E Yuan, Ji Yuan and people source MAVS in goose fibroblast inducing interferon stimulated gene OASL, Fig. 5 (D) is the expression of E Yuan, Ji Yuan and people source MAVS in goose fibroblast inducing interferon stimulated gene Viperin；

Fig. 6 (A) is E Yuan, Ji Yuan, people source MAVS the interferon hulFN- β in people/chicken cell induces HEK-293T cells Promoter activity, Fig. 6 (B) is that E Yuan, Ji Yuan, people source the MAVS interferon in people/chicken cell induces HEK-293T cells pierce Swash the promoter activity of response element hulSRE, Fig. 6 (C) is that E Yuan, Ji Yuan, people source MAVS induce HEK- in people/chicken cell The expression of interferon-stimulated gene huMX1 in 293T cells, Fig. 6 (D) are E Yuan, Ji Yuan, people source MAVS in people/chicken cell It is E Yuan, Ji Yuan, people source MAVS to induce the expression of interferon-stimulated gene hulFIT1 in HEK-293T cells, Fig. 6 (E) The promoter activity of interferon chlFN- β in people/chicken cell induction DF-1 cells, Fig. 6 (F) is E Yuan, Ji Yuan, people source MAVS The promoter activity of interferon controlling gene chlRF-7 in people/chicken cell induction DF-1 cells；

Fig. 7 (A) is wild type, missing CARD, missing TM and the table for lacking interferon-stimulated gene IFITM3 in PRR plasmids Up to level, Fig. 7 (B) is wild type, missing CARD, missing TM and the expression water for lacking interferon-stimulated gene MX in PRR plasmids Flat, Fig. 7 (C) is wild type, missing CARD, lacks TM and lack the expression of interferon-stimulated gene OASL in PRR plasmids, Fig. 7 (D) be wild type, missing CARD, missing TM and lack PRR plasmids in interferon-stimulated gene Viperin expression, Fig. 7 (E) is the expression of interferon-stimulated gene IFITM3 in wild type, deletion construct domain A and deletion construct domain B plasmids, Fig. 7 (F) is the expression of interferon-stimulated gene MX in wild type, deletion construct domain A and deletion construct domain B plasmids, Fig. 7 (G) expression for being interferon-stimulated gene OASL in wild type, deletion construct domain A and deletion construct domain B plasmids, Fig. 7 (H) For the expression of interferon-stimulated gene Viperin in wild type, deletion construct domain A and deletion construct domain B plasmids；

Fig. 8 (A) is to be overexpressed goose source MAVS to activate interferon access that NDV is inhibited to replicate, MAVS expression quantity, and Fig. 8 (B) was Expressing goose source MAVS activation interferon accesses inhibits NDV to replicate, and virus N P expression quantity, Fig. 8 (C) is to be overexpressed goose source MAVS activation Interferon access inhibits NDV to replicate, the expression quantity of interferon-stimulated gene IFITM3, and Fig. 8 (D) is to be overexpressed goose source MAVS activation Interferon access inhibits NDV to replicate, the expression quantity of interferon-stimulated gene MX, and Fig. 8 (E) is to be overexpressed goose source MAVS activation interference Plain access inhibits NDV to replicate, the expression quantity of interferon-stimulated gene OASL, and Fig. 8 (F) is to be overexpressed goose source MAVS to activate interferon Access inhibits NDV to replicate, the expression quantity of interferon-stimulated gene Viperin.

Specific implementation mode

The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright , the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The determination of 1 goose source MAVS gene orders of embodiment

According to the genome of existing goose on GenBank and the MAVS sequences of goose MAVS predictions, overlap is designed PCR amplification primer P1, P2, P3, P4 expand the MAVS genes of goose by the method for fusion DNA vaccine, and wherein P1, P2 are first round PCR Upstream and downstream primer, P3, P4 be second wheel PCR upstream and downstream primer, two-wheeled PCR product mixing after using P1 and P4 progress third Wheel PCR obtains full length sequence, and connection carrier T sequencing, primer P5, P6 are the fluorescence quantitative PCR detection sequence of MAVS sequences, specifically As shown in table 1 below：

Table 1

According to the form below 2 prepares PCR amplification system, and two-wheeled is divided to carry out over-lap PCR amplification, specific as follows：

Table 2

Specific response procedures are as shown in table 3 below：

Table 3

The first round expands：It is primer amplification MAVS genes front 1200bp or so piece with P1, P2 using GEF cDNA as template Section, is the segment of primer amplification MAVS genes rear portion 1800bp or so with P3, P4, and the segment of amplification is carried out respectively at 10% gel Agarose gel electrophoresis utilizes Tiangeng gel reclaims kit recovery purifying.

Second wheel amplification：Using two bar segments of first round amplification recycling as template, it is primer with P1, P4, expands MAVS bases The full length sequence of cause separates and recovers size by agarose gel electrophoresis and is the band of 2019bp, and purified.

By obtained MAVS segments link pGEM-T carriers (being purchased from Promega) conversion DH5 α (Escherichia coli), picking 12 A clone is identified with PCR obtains positive colony, and Shanghai Sangon Biotech Company is sent to be sequenced.Positive colony nucleotide sequence such as SEQ ID Shown in NO.7；Amino acid sequence is as shown in SEQ ID NO.6.

Jiang Eyuan, Ji Yuan, Ya Yuan, people source and the MAVS sequences in mouse source are compared, and the results are shown in Figure 1, goose source MAVS The homology of gene and duck source MAVS genes only has 55.8%, goose source MAVS is compared with NCBI forecasting sequences, as a result such as Shown in Fig. 2, homology is up to 96.2%, but wherein has the insertion of three sections of long sequences, and it is total to account for goose MAVS up to 828 bases for length The 41% of sequence length.

Goose MAVS and other species MAVS sequences are subjected to phylogenetic analysis, the results are shown in Figure 3：Goose MAVS and duck MAVS Affiliation is nearest, and the MAVS of various birds is classified as a branch for chadogram, and mammal MAVS affiliations are closer, quilt It is classified as a branch, primate is classified as a subbranch in mammal, and fish are individually for a branch.

Embodiment 2 builds goose source MAVS eukaryon expression plasmids

In order to build the total length expressed plasmids of goose MAVS of p3XFLAG-CMV-14 connections, with the overall length goose of carrier T connection MAVS is template, using P7 and P8 as primer amplification, after EcoR I/Xba I (being purchased from takara companies) digestion, is connected into P3XFLAG-CMV-14 expression vectors, specific as shown in table 4, wherein P7 and P8 are that structure wild type goose MAVS expression plasmids draw Object, P9 and P10 are structure missing PRR structural domain goose MAVS expression plasmid primers：

Table 4

Specific PCR system is as shown in table 5 below, specific as follows：

Table 5

Specific PCR conditions are as shown in table 6 below, specific as follows：

Table 6

The first round expands：It is that primer carries out PCR, the piece of amplification respectively with P7, P10 and P8, P9 using T-goMAVS as template Section, into row agarose gel electrophoresis, utilizes Tiangeng gel reclaims kit recovery purifying respectively at 10% gel.

Second wheel amplification：It is primer with P7, P8 using two bar segments of first round amplification recycling as template, amplification missing PRR The full length sequence of the goose MAVS genes of structural domain, is separated and recovered and is purified by agarose gel electrophoresis, through EcoR I/Xba I After (being purchased from takara companies) digestion, p3XFLAG-CMV-14 expression vectors (being purchased from Sigma companies) are connected into, conversion DH5 α are (big Enterobacteria), 6 clones of picking are identified with PCR obtains positive colony, and Shanghai Sangon Biotech Company is sent to be sequenced.

It is built using same method and lacks CARD, TM, the eukaryon expression plasmid of structural domain A and structural domain B are built Each Plasmid pattern figure is as shown in Figure 4.

3 external source of embodiment transfect goose MAVS can in goose cell induced high levels interferon-stimulated gene expression

Other than the MAVS eukaryon expression plasmids of structure goose source, researcher also constructs Ren Yuan and chicken source MAVS, in goose E Yuan, Ji Yuan and people source MAVS are overexpressed in primary fibroblast (GEF) respectively, is as follows：With endotoxin-free matter Grain extracts kit (being purchased from Tiangeng biochemical technology Co., Ltd) extraction p3XFLAG-CMV-14 empty carriers, goose MAVS, chicken MAVS With people's MAVS recombinant plasmids, transiently transfected by liposome (being purchased from life companies of the U.S.) transfection method after digestion identification is correct Cell sample is collected in GEF cells, transfection afterwards for 24 hours, extracts RNA, and inverted with promega M-MLV reverse transcription reagent box Record.

Obtained E Yuan, Ji Yuan and people source MAVS are utilized into fluorescence quantitative PCR detection interferon-stimulated gene IFITM3, The expression of MX, OASL and Viperin, as a result as shown in Fig. 5 (A)-Fig. 5 (D).

From Fig. 5 (A)-Fig. 5 (D) as can be seen that Real-time PCR (real-time quantitative PCR) the result shows that, chicken MAVS can The expression of the interferon-stimulated gene of 5-30 times of induction, the overexpression of people MAVS are only capable of the expression of low-level stimulation transcription factor, Under comparing, goose MAVS can induce the mRNA level in-site of the interferon-stimulated gene of highest level in GEF cells, from interference The expression of plain stimulated gene is capable of the I type interferon Pathway Activation situations of reacting cells.

Researcher again Jiang Eyuan, Ji Yuan and people source MAVS transfected to people source HEK-293T cells with above-mentioned same method and In the DF-1 cells of chicken source, using the expression of fluorescence quantitative PCR detection interferon-stimulated gene huMX1, hulFIT1, with double fluorescence The work of plain enzyme reporter gene detection technique detection hulFN- β-luc, hulSRE-luc, chlFN- β-luc and chlRF-7-luc Property, as a result as shown in Fig. 6 (A)-Fig. 6 (F).

From Fig. 6 (A)-Fig. 6 (F) as can be seen that the effect of MAVS has species specificity, i.e., in people's cell, people source MAVS Higher levels of interferon is generated than Ji Yuan and goose source MAVS；Similarly, chicken source MAVS can preferably stimulate I types in chicken cell Interferon access.

Embodiment 4 lacks influence of each structural domain to goose MAVS functions

MAVS albumen has several vital structural domains, is the areas CARD (active region) respectively, the areas TM (transmembrane region), The areas PRR (proline-rich region), respectively to the areas CARD, the insertion in the areas TM, the areas PRR and two sections of maximum length sequences (divides researcher It is not named as structural domain A and structural domain B) it is lacked, structure missing recombinant plasmid, the plasmid such as embodiment 2 of structure will be built Plasmid carry out functional verification, be as follows：It is (limited purchased from Tiangeng biochemical technology with endotoxin-free plasmid extracts kit Company) extraction recombinant plasmid, instantaneously turned by liposome (being purchased from life companies of the U.S.) transfection method after digestion identification is correct GEF cells are contaminated, cell sample is collected in transfection afterwards for 24 hours, extracts RNA, and inverted with promega M-MLV reverse transcription reagent box Then record utilizes the expression of fluorescence quantitative PCR detection interferon-stimulated gene IFITM3, MX, OASL and Viperin, knot Shown in fruit such as Fig. 7 (A)-Fig. 7 (D).

From Fig. 7 (A)-Fig. 7 (D) as can be seen that Real-time PCR the result shows that, with wild type MAVS overexpression group phases Than the areas missing CARD, the areas TM, structural domain A and structural domain B significantly inhibit the congenital immunity of goose MAVS activation, but lack PRR The scarce goose MAVS in area can induce the expression of higher levels of interferon-stimulated gene, illustrate that the areas PRR play negative regulation MAVS work( The effect of energy, the goose MAVS for lacking PRR can be used as a kind of better immunostimulant.

Embodiment 5 is overexpressed the influence that goose MAVS replicates NDV

The overexpression of goose MAVS can significantly activate I type interferon signal paths, the present embodiment great to have to aviculture The newcastle disease virus (NDV) of harm is model, and can verification goose MAVS play antiviral effect to NDV, be as follows：

(1) GEF cells are infected with NDV first, detects goose MAVS mRNA level in-sites in GEF cells, as a result shows that NDV cannot The mRNA level in-site of goose MAVS increases in induction GEF cells；

(2) by empty carrier, goose MAVS transfects GEF cells respectively, after transfection for 24 hours, is felt respectively with 1 infection multiplicity (MOI) NDV Herts33/56 are contaminated, collect cell sample after infection after 6,12,18,24 hours, extract RNA, and carry out reverse transcription；

(3) fluorescence quantitative PCR detection is carried out to target gene as template, as a result as shown in Fig. 8 (A)-Fig. 8 (F).

From Fig. 8 (A)-Fig. 8 (F) as can be seen that goose MAVS 1000-1500 times has been raised in transfection group, illustrate transfect at Work((Fig. 8 A)；Researcher has detected the mRNA that different infection time points are overexpressed viral NP protein in goose MAVS groups and control group As a result level shows that compared with the control group, transfection MAVS groups can significantly reduce virus N P eggs 6,12,18 hours after infection White mRNA level in-site illustrates that the prometaphase that goose MAVS can be infected in NDV significantly inhibits virus replication (Fig. 8 B)；Researcher is same When NDV is infected after the interferon-stimulated genes of different time points be detected, the results show that 6 after virus infects, 12 is small When, it is overexpressed the expression (Fig. 8 C-F) that goose MAVS significantly raises interferon-stimulated gene

As it can be seen that goose MAVS can be in virus infection early stage by activating I type interferon access suppressing virus replications.

In conclusion the present invention pass through expand goose source MAVS, it is determined that goose source MAVS genes, overall length 2019bp, find with NCBI forecasting sequence homologys are up to 96.2%, and verification finds that goose source MAVS and the goose source MAVS of missing PRR structural domains can make For potential goose immunostimulant and antiviral gene；Goose source MAVS can be lured in primary goose embryo fibroblast in the present invention The innate antiviral reaction of highest level is led, and the early stage of newcastle disease virus can be inhibited to replicate.

Applicant states that the present invention illustrates the process of the present invention, but the present invention not office by above-described embodiment It is limited to above-mentioned processing step, that is, does not mean that the present invention has to rely on above-mentioned processing step and could implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention Addition, the selection etc. of concrete mode, all fall within protection scope of the present invention and the open scope.

SEQUENCE LISTING

<110>China Agriculture Academe Shanghai Veterinary Institute (China Animal Health and Epidemiology Center Shanghai branch center)

<120>A kind of antiviral signal protein of goose source mitochondria and its application

<130> 2018

<160> 19

<170> PatentIn version 3.3

<210> 1

<211> 68

<212> PRT

<213>Artificial synthesized sequence

<400> 1

Gly His Ile Leu Arg Asn Met Asp Lys Phe Lys Asn Ile His Val Ala

1 5 10 15

Ser Leu Val Asp Ser Leu Ser Cys Leu Thr Glu Ala Asp Arg Asp Glu

20 25 30

Leu His Thr Arg Gly Glu Val Arg Gly Ser Asn Ala Thr Ala Tyr Lys

35 40 45

Leu Tyr Gln His Val Lys Cys Arg Arg Gly Trp Val Thr Asp Leu Ile

50 55 60

Asn Ala Leu His

65

<210> 2

<211> 114

<212> PRT

<213>Artificial synthesized sequence

<400> 2

Ser Ala Pro Pro Ala Ala Pro Asn Pro Arg Val Ala Ser Phe Pro Pro

1 5 10 15

Asp Ala Gln Pro Val Ser Ala Gln Leu Pro Phe Ser Gly Pro Lys Pro

20 25 30

Ala Ala Asp Ala Pro Leu Ala Asp Pro Pro Arg Cys Thr Ser Pro Thr

35 40 45

Gly Gly His Pro Pro Leu Gln Arg Ala Ala Ala Thr Ala Ala Ser Pro

50 55 60

Ala Gly Ser Ser Ala Ala Ser Val Asp Val Pro Ser Thr Asp Leu Asp

65 70 75 80

Ala Arg Ala Pro Val Gln Glu Lys Pro Leu Glu Gln Lys Ser Pro Gln

85 90 95

Pro Pro Leu Gln Pro Leu Ser Ala Ala Arg Ala Gly Ala Ser Asp Gly

100 105 110

Gln Ser

<210> 3

<211> 55

<212> PRT

<213>Artificial synthesized sequence

<400> 3

Ala Ala Gly Val Val Leu Pro Pro Ala Ala Pro Pro Glu Gln Gly Arg

1 5 10 15

Glu Trp Leu Gly Gln Arg Tyr Pro Val Cys Val Asp Asn Gly Cys Phe

20 25 30

Gly Asn Ala Lys His Leu Arg Arg Ala Ala Pro Gly Ser Gly Leu Gly

35 40 45

Thr Ala Val Pro Leu Arg Asp

50 55

<210> 4

<211> 196

<212> PRT

<213>Artificial synthesized sequence

<400> 4

Ser Val Gly Ala Ala Gly Arg Pro Thr Ser Pro Pro Gly Glu Phe Cys

1 5 10 15

His Lys His Leu Gly Leu Ser His His Pro Gly Gly Gly Arg Gly Ala

20 25 30

Gln Gln Ala Gly Arg Pro Pro Leu Arg Gly Gly Arg Gln Pro Ala Gly

35 40 45

Ser Arg Pro Ala Pro Gly Arg Leu Arg Phe Arg Gly His Arg Ser Pro

50 55 60

Gly Leu Glu Glu Arg Pro Ala Pro Ala Glu His Arg Gln Leu Arg Ser

65 70 75 80

Arg Arg Ser Thr Arg Gly Ile Arg Leu Gly Thr Leu Leu Leu Gly Ile

85 90 95

Leu Arg Arg Arg Ser Gly Gly Ser Thr Leu Leu Val Pro Pro Glu Leu

100 105 110

Glu Asn Arg Arg Asp Pro Arg Gly Ser Arg Pro Gln Arg Pro Ala Glu

115 120 125

Gly Glu Gln Arg Arg Pro Gly Arg Arg Ser Arg Ser Leu Gly Arg Pro

130 135 140

Pro Gly Phe Arg Leu Glu Arg Arg Gln Arg Arg His Ala Trp Gln Arg

145 150 155 160

Pro Gln Pro Trp Gly Gln Pro Arg Ala Val Pro Ala Leu Pro Arg Pro

165 170 175

Gly Arg Gly His Arg Ala His Leu Arg Arg Gly Val Pro Gly Val Arg

180 185 190

Pro Ala Ala Glu

195

<210> 5

<211> 19

<212> PRT

<213>Artificial synthesized sequence

<400> 5

Ala Ala Ala Ala Pro Arg Glu Val Pro Leu Pro Gly Arg Asn Pro Val

1 5 10 15

Trp Arg Val

<210> 6

<211> 672

<212> PRT

<213>Artificial synthesized sequence

<400> 6

Met Gly Phe Ala Glu Asp Lys Val Tyr Gly His Ile Leu Arg Asn Met

1 5 10 15

Asp Lys Phe Lys Asn Ile His Val Ala Ser Leu Val Asp Ser Leu Ser

20 25 30

Cys Leu Thr Glu Ala Asp Arg Asp Glu Leu His Thr Arg Gly Glu Val

35 40 45

Arg Gly Ser Asn Ala Thr Ala Tyr Lys Leu Tyr Gln His Val Lys Cys

50 55 60

Arg Arg Gly Trp Val Thr Asp Leu Ile Asn Ala Leu His Gln Asn Asn

65 70 75 80

Ala Gly His Leu Ala Glu Glu Leu Gln Gln Val Tyr Asp Leu Tyr Gln

85 90 95

Thr Pro Pro Pro Thr Ala Ser Ala Pro Pro Ala Ala Pro Asn Pro Arg

100 105 110

Val Ala Ser Phe Pro Pro Asp Ala Gln Pro Val Ser Ala Gln Leu Pro

115 120 125

Phe Ser Gly Pro Lys Pro Ala Ala Asp Ala Pro Leu Ala Asp Pro Pro

130 135 140

Arg Cys Thr Ser Pro Thr Gly Gly His Pro Pro Leu Gln Arg Ala Ala

145 150 155 160

Ala Thr Ala Ala Ser Pro Ala Gly Ser Ser Ala Ala Ser Val Asp Val

165 170 175

Pro Ser Thr Asp Leu Asp Ala Arg Ala Pro Val Gln Glu Lys Pro Leu

180 185 190

Glu Gln Lys Ser Pro Gln Pro Pro Leu Gln Pro Leu Ser Ala Ala Arg

195 200 205

Ala Gly Ala Ser Asp Gly Gln Ser Ala Arg Gly Pro Leu Pro Cys Pro

210 215 220

Ser Glu Ala Gln Gln Ala Ala Ala Gly Pro Pro Gly Ala Ala Gly Val

225 230 235 240

Val Leu Pro Pro Ala Ala Pro Pro Glu Gln Gly Arg Glu Trp Leu Gly

245 250 255

Gln Arg Tyr Pro Val Cys Val Asp Asn Gly Cys Phe Gly Asn Ala Lys

260 265 270

His Leu Arg Arg Ala Ala Pro Gly Ser Gly Leu Gly Thr Ala Val Pro

275 280 285

Leu Arg Asp Pro Val Thr Ala Arg Gly Ala Gly Gln Pro Arg Asn Glu

290 295 300

Pro Gln Glu Asp Val Tyr Val Ser Thr Glu Leu Pro Ala Gly Leu Ala

305 310 315 320

Glu Pro Leu Cys Gly Glu Gly Val Pro Gln Pro Arg Gly Pro Gln Gly

325 330 335

Thr Gln Glu Lys Gln Ala Glu Leu Pro Glu His Arg Gly Pro Pro Ser

340 345 350

Ser Phe Val Asp Val Arg Ser Pro Leu Leu Ile Gln Gln Gln Phe Asp

355 360 365

Ala Glu Gln Lys Arg Val Gly Thr Leu Arg Glu His Glu Glu Gly Ala

370 375 380

Thr Arg Met Glu Thr Ile Pro Pro Ala Ala Ala Pro Arg Asp Thr Ser

385 390 395 400

Pro Ser Arg Asp Val Ser Trp Lys Leu Pro Val Pro Glu Glu Ala Leu

405 410 415

Pro Ala Gly Lys Ala Ala Gly Ser Thr Pro Ser Val Pro Ala Glu Glu

420 425 430

Lys Val Leu Pro Ala Ser Ala Ala Pro Leu Gln Gly Pro Ala Val Gly

435 440 445

Gly Ser Ser Val Gly Ala Ala Gly Arg Pro Thr Ser Pro Pro Gly Glu

450 455 460

Phe Cys His Lys His Leu Gly Leu Ser His His Pro Gly Gly Gly Arg

465 470 475 480

Gly Ala Gln Gln Ala Gly Arg Pro Pro Leu Arg Gly Gly Arg Gln Pro

485 490 495

Ala Gly Ser Arg Pro Ala Pro Gly Arg Leu Arg Phe Arg Gly His Arg

500 505 510

Ser Pro Gly Leu Glu Glu Arg Pro Ala Pro Ala Glu His Arg Gln Leu

515 520 525

Arg Ser Arg Arg Ser Thr Arg Gly Ile Arg Leu Gly Thr Leu Leu Leu

530 535 540

Gly Ile Leu Arg Arg Arg Ser Gly Gly Ser Thr Leu Leu Val Pro Pro

545 550 555 560

Glu Leu Glu Asn Arg Arg Asp Pro Arg Gly Ser Arg Pro Gln Arg Pro

565 570 575

Ala Glu Gly Glu Gln Arg Arg Pro Gly Arg Arg Ser Arg Ser Leu Gly

580 585 590

Arg Pro Pro Gly Phe Arg Leu Glu Arg Arg Gln Arg Arg His Ala Trp

595 600 605

Gln Arg Pro Gln Pro Trp Gly Gln Pro Arg Ala Val Pro Ala Leu Pro

610 615 620

Arg Pro Gly Arg Gly His Arg Ala His Leu Arg Arg Gly Val Pro Gly

625 630 635 640

Val Arg Pro Ala Ala Glu Ile Ala Glu Ser Asn Gly Gly Ala Ala Ala

645 650 655

Ala Pro Arg Glu Val Pro Leu Pro Gly Arg Asn Pro Val Trp Arg Val

660 665 670

<210> 7

<211> 2019

<212> DNA

<213>Artificial synthesized sequence

<400> 7

atgggctttg cggaggacaa ggtgtacggc cacatcctga ggaacatgga caagttcaag 60

aacatccacg tggcgtcgct ggtcgattcc ctcagctgcc tcaccgaagc cgacagggac 120

gaacttcata cccgagggga ggtacggggc agcaatgcaa ccgcctacaa gttatatcag 180

cacgtgaagt gccgccgggg ctgggtgacc gacctgatca acgcgctgca ccagaacaac 240

gcggggcacc tggccgaaga gctgcagcag gtctatgacc tctaccaaac ccctcctcca 300

acagcctcag ctccaccagc tgctccaaat cctcgtgtgg cctccttccc tcctgatgcc 360

cagcccgtca gtgcccagct gccattctcg ggccccaagc ccgctgcgga cgccccgctg 420

gccgatcctc cccgctgcac ctcgcccacc ggcggccatc cacctctcca gcgcgctgct 480

gccaccgctg cgagcccagc cgggagctca gctgcgagcg tggacgttcc cagcaccgac 540

ctggacgcca gggcccctgt gcaggagaag cccctggaac aaaagagtcc ccagccaccg 600

ctgcagccgc tgagcgcggc ccgtgctgga gcgagcgacg ggcagagcgc gcgggggccg 660

ctcccgtgcc cctccgaggc tcagcaggcg gcagcggggc ccccgggggc tgccggcgtg 720

gttttgccgc cggctgctcc ccccgagcag ggccgggagt ggctgggcca gcgctacccg 780

gtgtgcgtgg acaacgggtg cttcgggaac gccaagcacc tgcgccgcgc cgcgccgggc 840

tcggggctgg gcacggccgt cccgctgagg gacccggtca ccgctcgtgg cgccgggcag 900

cccaggaacg agccccagga agacgtctac gtctccaccg agttgcccgc ggggttggca 960

gagcctctgt gcggcgaggg ggtgccgcag ccccgcggcc cgcaggggac gcaggaaaag 1020

caggcggagc tccccgagca ccgagggcct ccgagcagct tcgtggacgt gcgcagcccc 1080

ctcctcatac agcagcagtt tgacgcggag cagaagcggg tcgggacgct gcgagagcac 1140

gaagagggag ccacccggat ggaaacaatc cccccggccg ccgcccccag agacacttcc 1200

ccatcccgcg acgtctcctg gaagcttccc gtgccagagg aagcgctgcc tgcggggaag 1260

gcggccggca gcaccccctc cgtgccggca gaggagaaag tgctcccggc ctcggcggcc 1320

cctctccagg gcccagcggt cgggggaagc tctgtgggcg cagctgggag gccgacgtcc 1380

cccccggggg agttctgcca caagcatctg ggcctctccc accatcccgg aggaggacgc 1440

ggagctcagc aagccgggcg tcctcctctc cgtggcgggc gacagcccgc aggcagccgc 1500

ccggcccccg ggcggctccg cttccgaggc caccgatcac ctgggcttga ggagcgaccc 1560

gctcctgctg agcaccgaca gctcaggtcc cggaggagca cgcgagggat ccgcctcggg 1620

acgttgctcc tcggcatcct gcggagacga agcgggggga gcacgctcct ggtccccccc 1680

gagctggaga accgacgaga tccgcgtggc tcacggcccc agcgccctgc tgaaggcgag 1740

caacgacgtc ccggacgacg gagccggtcc ctcgggcggc cccccggctt ccgcctcgag 1800

cggaggcagc gacgccacgc ctggcagcgt ccccagccct ggggacagcc ccgggccgtc 1860

cctgccctac ctcgtcccgg ccgtgggcat cgcgctcatc tccgccgtgg cgttcctggt 1920

gtacgcccgg ctgcagaaat agcggagagc aacgggggtg ccgcggccgc cccgcgggag 1980

gttcctcttc ccggcaggaa tcccgtctgg cgcgtttga 2019

<210> 8

<211> 558

<212> PRT

<213>Artificial synthesized sequence

<400> 8

Met Gly Phe Ala Glu Asp Lys Val Tyr Gly His Ile Leu Arg Asn Met

1 5 10 15

Asp Lys Phe Lys Asn Ile His Val Ala Ser Leu Val Asp Ser Leu Ser

20 25 30

Cys Leu Thr Glu Ala Asp Arg Asp Glu Leu His Thr Arg Gly Glu Val

35 40 45

Arg Gly Ser Asn Ala Thr Ala Tyr Lys Leu Tyr Gln His Val Lys Cys

50 55 60

Arg Arg Gly Trp Val Thr Asp Leu Ile Asn Ala Leu His Gln Asn Asn

65 70 75 80

Ala Gly His Leu Ala Glu Glu Leu Gln Gln Val Tyr Asp Leu Tyr Gln

85 90 95

Thr Pro Pro Pro Thr Ala Ala Arg Gly Pro Leu Pro Cys Pro Ser Glu

100 105 110

Ala Gln Gln Ala Ala Ala Gly Pro Pro Gly Ala Ala Gly Val Val Leu

115 120 125

Pro Pro Ala Ala Pro Pro Glu Gln Gly Arg Glu Trp Leu Gly Gln Arg

130 135 140

Tyr Pro Val Cys Val Asp Asn Gly Cys Phe Gly Asn Ala Lys His Leu

145 150 155 160

Arg Arg Ala Ala Pro Gly Ser Gly Leu Gly Thr Ala Val Pro Leu Arg

165 170 175

Asp Pro Val Thr Ala Arg Gly Ala Gly Gln Pro Arg Asn Glu Pro Gln

180 185 190

Glu Asp Val Tyr Val Ser Thr Glu Leu Pro Ala Gly Leu Ala Glu Pro

195 200 205

Leu Cys Gly Glu Gly Val Pro Gln Pro Arg Gly Pro Gln Gly Thr Gln

210 215 220

Glu Lys Gln Ala Glu Leu Pro Glu His Arg Gly Pro Pro Ser Ser Phe

225 230 235 240

Val Asp Val Arg Ser Pro Leu Leu Ile Gln Gln Gln Phe Asp Ala Glu

245 250 255

Gln Lys Arg Val Gly Thr Leu Arg Glu His Glu Glu Gly Ala Thr Arg

260 265 270

Met Glu Thr Ile Pro Pro Ala Ala Ala Pro Arg Asp Thr Ser Pro Ser

275 280 285

Arg Asp Val Ser Trp Lys Leu Pro Val Pro Glu Glu Ala Leu Pro Ala

290 295 300

Gly Lys Ala Ala Gly Ser Thr Pro Ser Val Pro Ala Glu Glu Lys Val

305 310 315 320

Leu Pro Ala Ser Ala Ala Pro Leu Gln Gly Pro Ala Val Gly Gly Ser

325 330 335

Ser Val Gly Ala Ala Gly Arg Pro Thr Ser Pro Pro Gly Glu Phe Cys

340 345 350

His Lys His Leu Gly Leu Ser His His Pro Gly Gly Gly Arg Gly Ala

355 360 365

Gln Gln Ala Gly Arg Pro Pro Leu Arg Gly Gly Arg Gln Pro Ala Gly

370 375 380

Ser Arg Pro Ala Pro Gly Arg Leu Arg Phe Arg Gly His Arg Ser Pro

385 390 395 400

Gly Leu Glu Glu Arg Pro Ala Pro Ala Glu His Arg Gln Leu Arg Ser

405 410 415

Arg Arg Ser Thr Arg Gly Ile Arg Leu Gly Thr Leu Leu Leu Gly Ile

420 425 430

Leu Arg Arg Arg Ser Gly Gly Ser Thr Leu Leu Val Pro Pro Glu Leu

435 440 445

Glu Asn Arg Arg Asp Pro Arg Gly Ser Arg Pro Gln Arg Pro Ala Glu

450 455 460

Gly Glu Gln Arg Arg Pro Gly Arg Arg Ser Arg Ser Leu Gly Arg Pro

465 470 475 480

Pro Gly Phe Arg Leu Glu Arg Arg Gln Arg Arg His Ala Trp Gln Arg

485 490 495

Pro Gln Pro Trp Gly Gln Pro Arg Ala Val Pro Ala Leu Pro Arg Pro

500 505 510

Gly Arg Gly His Arg Ala His Leu Arg Arg Gly Val Pro Gly Val Arg

515 520 525

Pro Ala Ala Glu Ile Ala Glu Ser Asn Gly Gly Ala Ala Ala Ala Pro

530 535 540

Arg Glu Val Pro Leu Pro Gly Arg Asn Pro Val Trp Arg Val

545 550 555

<210> 9

<211> 1677

<212> DNA

<213>Artificial synthesized sequence

<400> 9

atgggctttg cggaggacaa ggtgtacggc cacatcctga ggaacatgga caagttcaag 60

aacatccacg tggcgtcgct ggtcgattcc ctcagctgcc tcaccgaagc cgacagggac 120

gaacttcata cccgagggga ggtacggggc agcaatgcaa ccgcctacaa gttatatcag 180

cacgtgaagt gccgccgggg ctgggtgacc gacctgatca acgcgctgca ccagaacaac 240

gcggggcacc tggccgaaga gctgcagcag gtctatgacc tctaccaaac ccctcctcca 300

acagccgcgc gggggccgct cccgtgcccc tccgaggctc agcaggcggc agcggggccc 360

ccgggggctg ccggcgtggt tttgccgccg gctgctcccc ccgagcaggg ccgggagtgg 420

ctgggccagc gctacccggt gtgcgtggac aacgggtgct tcgggaacgc caagcacctg 480

cgccgcgccg cgccgggctc ggggctgggc acggccgtcc cgctgaggga cccggtcacc 540

gctcgtggcg ccgggcagcc caggaacgag ccccaggaag acgtctacgt ctccaccgag 600

ttgcccgcgg ggttggcaga gcctctgtgc ggcgaggggg tgccgcagcc ccgcggcccg 660

caggggacgc aggaaaagca ggcggagctc cccgagcacc gagggcctcc gagcagcttc 720

gtggacgtgc gcagccccct cctcatacag cagcagtttg acgcggagca gaagcgggtc 780

gggacgctgc gagagcacga agagggagcc acccggatgg aaacaatccc cccggccgcc 840

gcccccagag acacttcccc atcccgcgac gtctcctgga agcttcccgt gccagaggaa 900

gcgctgcctg cggggaaggc ggccggcagc accccctccg tgccggcaga ggagaaagtg 960

ctcccggcct cggcggcccc tctccagggc ccagcggtcg ggggaagctc tgtgggcgca 1020

gctgggaggc cgacgtcccc cccgggggag ttctgccaca agcatctggg cctctcccac 1080

catcccggag gaggacgcgg agctcagcaa gccgggcgtc ctcctctccg tggcgggcga 1140

cagcccgcag gcagccgccc ggcccccggg cggctccgct tccgaggcca ccgatcacct 1200

gggcttgagg agcgacccgc tcctgctgag caccgacagc tcaggtcccg gaggagcacg 1260

cgagggatcc gcctcgggac gttgctcctc ggcatcctgc ggagacgaag cggggggagc 1320

acgctcctgg tcccccccga gctggagaac cgacgagatc cgcgtggctc acggccccag 1380

cgccctgctg aaggcgagca acgacgtccc ggacgacgga gccggtccct cgggcggccc 1440

cccggcttcc gcctcgagcg gaggcagcga cgccacgcct ggcagcgtcc ccagccctgg 1500

ggacagcccc gggccgtccc tgccctacct cgtcccggcc gtgggcatcg cgctcatctc 1560

cgccgtggcg ttcctggtgt acgcccggct gcagaaatag cggagagcaa cgggggtgcc 1620

gcggccgccc cgcgggaggt tcctcttccc ggcaggaatc ccgtctggcg cgtttga 1677

<210> 10

<211> 21

<212> DNA

<213>Artificial synthesized sequence

<400> 10

atgggctttg cggaggacaa g 21

<210> 11

<211> 30

<212> DNA

<213>Artificial synthesized sequence

<400> 11

ctggcacggg aagcttccag gagacgtcgc 30

<210> 12

<211> 30

<212> DNA

<213>Artificial synthesized sequence

<400> 12

gcgacgtctc ctggaagctt cccgtgccag 30

<210> 13

<211> 24

<212> DNA

<213>Artificial synthesized sequence

<400> 13

tcaaacgcgc cagacgggat tcct 24

<210> 14

<211> 19

<212> DNA

<213>Artificial synthesized sequence

<400> 14

gccacatcct gaggaacat 19

<210> 15

<211> 21

<212> DNA

<213>Artificial synthesized sequence

<400> 15

ggtatgaagt tcgtccctgt c 21

<210> 16

<211> 31

<212> DNA

<213>Artificial synthesized sequence

<400> 16

ccggaattca tgggctttgc ggaggacaag g 31

<210> 17

<211> 30

<212> DNA

<213>Artificial synthesized sequence

<400> 17

tgctctagaa acgcgccaga cgggattcct 30

<210> 18

<211> 30

<212> DNA

<213>Artificial synthesized sequence

<400> 18

cctcctccaa cagccgcgcg ggggccgctc 30

<210> 19

<211> 30

<212> DNA

<213>Artificial synthesized sequence

<400> 19

gagcggcccc cgcgcggctg ttggaggagg 30

Claims (10)

1. a kind of antiviral signal protein of goose source mitochondria, which is characterized in that the signal protein includes active region, proline In enrichment region, structural domain A, structural domain B or transmembrane region any one or at least two combination.

2. the antiviral signal protein of goose source according to claim 1 mitochondria, which is characterized in that the ammonia of the active region Base acid sequence is as shown in SEQ ID NO.1；

Preferably, the amino acid sequence of the proline-rich region is as shown in SEQ ID NO.2；

Preferably, the amino acid sequence of the structural domain A is as shown in SEQ ID NO.3；

Preferably, the amino acid sequence of the structural domain B is as shown in SEQ ID NO.4；

Preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO.5.

3. the antiviral signal protein of goose source according to claim 1 or 2 mitochondria, which is characterized in that the signal protein Including active region, proline-rich region, structural domain A, structural domain B and transmembrane region；

Preferably, the amino acid sequence of the signal protein is as shown in SEQ ID NO.6, nucleotide sequence such as SEQ ID Shown in NO.7；

Preferably, the signal protein includes active region, structural domain A, structural domain B and transmembrane region；

Preferably, the amino acid sequence of the signal protein is as shown in SEQ ID NO.8, nucleotide sequence such as SEQ ID Shown in NO.9.

4. a kind of preparation method of the antiviral signal protein of goose source as claimed in any one of claims 1-3 mitochondria, special Sign is, includes the following steps：Using the genome of existing goose on GenBank and the MAVS sequences of goose MAVS predictions as mould Plate is designed amplimer, is expanded using the method for over-lap PCR；

Preferably, the nucleotide sequence of the primer is as shown in SEQ ID NO.10-13.

5. encoding the gene of the antiviral signal protein of goose source as claimed in any one of claims 1-3 mitochondria.

6. a kind of recombinant plasmid, which is characterized in that it includes the genes described in claim 5.

7. a kind of recombinant virus, which is characterized in that it includes the genes described in claim 5.

8. a kind of vaccine adjuvant, which is characterized in that include the recombinant plasmid or as claimed in claim 7 described in claim 6 Recombinant virus.

9. a kind of host cell, which is characterized in that it includes the recombinant plasmids described in claim 6；

Preferably, the host cell is Escherichia coli.

10. gene as claimed in claim 5, recombinant plasmid as claimed in claim 6, recombination as claimed in claim 7 Virus or vaccine adjuvant as claimed in claim 8 are used to prepare antiviral and/or immune activation drug；

Preferably, the virus is newcastle disease virus.