CN108752481A - Merge the preparation and application of antibacterial peptide VASP biological agents in Novel pig source - Google Patents

Abstract

The invention discloses preparations and application that antibacterial peptide VASP biological agents are merged in Novel pig source.Fusion pig antibacterial peptide VASP provided by the present invention is following A1), A2) or A3)：A1) amino acid sequence is the protein of sequence 1 in sequence table；A2) amino acid sequence shown in sequence in sequence table 2 by the substitution of one or several amino acid residues and/or is lacked and ored add and protein with the same function；A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label.Experiments have shown that, the VASP of the present invention can promote lymphopoiesis and quantity of leucocyte to increase, inhibit the growth of pathogenic microorganisms, enhancing humoral immunity (IgG, IgG1, IgG2a) and cellular immunity (CD4+, CD8+T cell), the immunoregulation capability of raising animal and anti-infective survival rate.

Description

Merge the preparation and application of antibacterial peptide VASP biological agents in Novel pig source

Technical field

The invention belongs to biotechnologys and genetic engineering field, are related to Novel pig source fusion antibacterial peptide VASP biological agents It prepares and applies.

Background technology

The main problem that limitation aquaculture industry of China further develops and increases economic efficiency now is prevention and control Animal diseases, carries The development and production of high animal product quality and high-quality cheap feed.China's export animal product is because of quarantine and the residual problem of medicine, outlet Proportion is very low, if pork is more than 230,000 tons, only accounts for 4% or so of the quantum of international trade.The especially beans of Sichuan animal feed The 70% of the energy feeds raw material such as 90% and corn of the protein raw materials such as the dregs of rice, which all relies on, to be externally supplied, this just seriously constrains me The raising of the Animal husbandry production development and economic benefit of state.Therefore, how to reduce the use of antibiosis class element additive in feed, gram Take the residual harm of medicine of animal product, effective prevention and control Animal diseases occur, ensure the production of the organic animal foodstuff of green, the protection people Masses' life and health safety, it has also become the great science and technology and social economy's problem of contemporary urgent need to resolve.

Current China's livestock and poultry breeding industry is improved with intensive degree, and livestock and poultry cultivation scale and cultivation density are growing, It is strong under the pressure of the pressure of control more than 30 kinds of infectiousness cause of disease transmission of infection diffusion, especially bird flu and breathing breeding syndrome etc. Sick successive cycle outburst；Diarrhea caused by drug-fast bacteria in animal-breeding, sramana, Escherichia coli, streptococcus, blue otopathy (PRRSV), Various bacteriums, the viral diseases such as circovirus (PCV2), swine fever (CSFV), the serious development for hindering animal husbandry；Also make antibiosis The medicated premixes such as element are very serious in the abuse condition of feed, and the pollution problem of livestock and poultry and antibiotic feed additive is Bottleneck through being improved as restriction China's livestock and poultry breeding industry development level and economic benefit.The abuse of fodder antibiotics additive is not Feeding cost is only improved, also causes the drug resistance of pathogenic microorganism and pathogenicity to be remarkably reinforced, seriously destroys animal alimentary canal Microecological balance, remain and be enriched in animal body after long-time service, livestock and poultry body weakens health because of poisonous side effect of medicine Level, immune disease-resistance power are remarkably decreased.Such vicious circle, the various strong sexually transmitted diseases of livestock and poultry frequently occur, it is difficult to control and Treatment.And on the other hand, the animal products food-safety problem of medicament residue and getting worse not only directly threatens the mankind certainly The health of body also counteracts the development of aquaculture.

Animal feed adds antibiotic, and induction germ not only drug resistance is occurred by long-term or inappropriate use, but also dynamic There is medicament residue in produce product, cause serious food safety hazards, damage human health and incur the medicine for the treatment of antibiotic Object fails.The developed regions such as European Union, the U.S. start to limit antibiotic for 2006 comprehensively as feed addictive.China also will be stringent It limits feed and adds antibiotic.Modern farming is badly in need of the noresidue of exploitation substitution conventional antibiotic, non-resistant induction, safe nothing The new bio feed and its additive of pollution.

Antibacterial peptide molecular weight is small, isolates and purifies and examine that there are certain difficulties.Antibacterial peptide in scientific research at present and application Main source is chemical synthesis, but chemical synthesis is of high cost, and amount is small, is unfavorable for large-scale application.And existing single antibacterial Peptide molecule there are antimicrobial spectrums it is relatively narrow, bioactivity is relatively low the shortcomings of；Using genetic engineering means, recombination connects multiple antibacterial peptidyls It is anti-to obtain the efficient recombination with the anti-multiple-microorganism of wide spectrum (G+, G-, virus, fungi, parasite) and immunoregulatory activity for cause Bacterium peptide molecule is to overcome the prior art and the important new way of product defects.

Also gradually increase in relation to antibacterial peptide gene and other related gene fusion clonings and the report of expression, but rarely found has Close the small peptide Gene Fusion of a variety of antibacterials and immunoregulatory activity, effectively form a variety of anti-infection activity molecules after coexpression and Cooperate with report that is anti-infective and improving immune level.

Invention content

A purpose of the invention is to provide Novel pig source fusion antibacterial peptide VASP biological agents.

A kind of protein (VASP) provided by the invention, be following a)-e) in any protein：

A) amino acid sequence includes the protein of amino acid sequence shown in sequence 1 in sequence table；

B) amino acid sequence amino acid residue shown in sequence in sequence table 1 forms；

C) by substitution and/or missing of the amino acid sequence by one or several amino acid residues defined by a) or b) And/or it adds and there is the protein for improving animal immune ability function；

D) amino acid sequence defined by and a) or b) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology of person and the protein with raising animal immune ability function；

E) a)-d) in it is any defined by protein N-terminal and/or C-terminal connection label after obtained fusion protein.

The nucleic acid molecules for encoding above-mentioned albumen are also the scope of protection of the invention.

Above-mentioned nucleic acid molecules are following 1) -4) in it is any shown in nucleic acid molecules：

1) its coded sequence includes sequence 2 in sequence table；

2) its coded sequence is sequence 2 in sequence table；

1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and the DNA for encoding albumen described in claim 1 divides Son；

1) or 2) 4) with the DNA molecular that limits with 80% or more or 90% or more homology and the above-mentioned albumen of coding DNA molecular.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.VASP shown in 2 coded sequence 1 of sequence.

Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side Method is mutated the nucleotide sequence of the coding VASP of the present invention.Those have and are detached with the present invention by manually modified The nucleotide sequence 75% of obtained VASP or the nucleotide of higher homogeneity, as long as encoding VASP and having the function of VASP, It is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.

Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair Shown in bright coded sequence 2 amino acid sequence form protein nucleotide sequence have 75% higher or 85% or Higher or 90% higher or 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or computer software It is evaluated.Using computer software, homogeneity between two or more sequences can use percentage (%) to indicate, can be with For evaluating the homogeneity between correlated series.

In above application, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min； Or, in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, hybridizes under the conditions of 65 DEG C and wash film.

Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90% or 95% or more homogeneity.

In above application, B2) described in the nucleic acid molecules containing coding VASP expression cassette (VASP expression casettes), be Refer to express the DNA of VASP in host cell, which not only may include the promoter for starting VASP genetic transcriptions, may be used also Terminator including terminating VASP genetic transcriptions.Further, the expression cassette may also include enhancer sequence.

The recombinant vector of the VASP expression casettes can be contained with existing vector construction.

In above application, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid is concretely PVAX1 carriers.

B3) recombinant vector contains the DNA sequence dna for encoding VASP shown in sequence 2.Recombination described further Carrier concretely pVAX1.The pVAX1-SP is the DNA fragmentation identified the Hind III and Xbal of pVAX1 carriers between sequence Replace with VASP genes shown in sequence 1, obtained recombinant vector.

In one embodiment of the invention, the recombination eukaryotic vector is that will be obtained in the VASP channel genes pVAX1 The recombinant plasmid arrived.

In above application, the transgenic cell line does not include propagating materials.

Following 1) -3) any one of biomaterial is also the scope of protection of the invention：

1) contain the expression cassette of above-mentioned nucleic acid molecules；

2) contain the recombinant vector of above-mentioned nucleic acid molecules；

3) contain the recombinant bacterium or transgenic cell line of above-mentioned nucleic acid molecules；

4) contain the nano particle of the recombinant vector.

Above-mentioned nano particle is the nano particle for wrapping up recombinant vector chitosan or cationic-liposome.

The application of above-mentioned protein or above-mentioned nucleic acid molecules or above-mentioned biomaterial in following C1 or C2 or C3 is also In the scope of protection of the invention：

C1, animal immune ability is being improved；

C2, it prepares and improves animal immune ability product；

C3, preparation act on the anti-infective product of wide spectrum of animal.

In above application, the raising animal immune ability is at least one of following M1-M5：

M1, the growth for inhibiting pathogenic microorganisms；

M2, the increase for promoting immunocyte；

M3, promote vaccine-induced immune response；

M4, promote cellular immunity and/or humoral immunity；

M5, animal development and growth-weight gain are improved；

And/or the pathogenic microorganisms is specially Escherichia coli, staphylococcus aureus, mycoplasma hyopneumoniae, pig breeding With disordered breathing syndrome virus or swine fever virus；

And/or the immunocyte is specially lymphocyte or leucocyte；

And/or the antibody specific is IgG, IgG1 and/or IgG2a；

And/or the wide spectrum it is anti-infective for antibacterium, antiviral, true antibacterial or anti parasitic.

Another object of the present invention is to provide following product.

The present invention provides following any products of X1 or X2：

X1, biological agent contain following X3a, X3b or X3c：

X3a, above-mentioned protein；

X3b, above-mentioned nucleic acid molecules；

X3c, above-mentioned biomaterial；

X2, the reagent set for improving animal immune ability, by above-mentioned X1 and antibiotic group at.

In the said goods, the biological agent can using X3a, X3b or X3c as active constituent, can also by X3a, X3b or X3c is active constituent with the composition that other substances that can improve animal immune ability are combined.

In the said goods, the antibiotic can be kanamycins.

3rd purpose of the invention is to provide a kind of method improving animal immune ability.

Method provided by the invention includes applying above-mentioned protein or above-mentioned nucleic acid molecules or above-mentioned biological material to animal Material or the biological agent or the reagent set, improve the immunocompetence of the animal.

Among the above, the animal is H1 or H2：

H1, mammal；

H2, mouse.

Among the above, the pathogenic microorganisms can be Escherichia coli, salmonella, staphylococcus aureus, streptococcus.

The immunocyte can be lymphocyte (such as CD4+ or CD8+), red blood cell or leucocyte.

The antibody can be IgG, IgG1 and/or IgG2a.The antibody specific can be the antibody of the pathogenic microorganisms.

In the present invention, the animal can be mammal.The mammal concretely mouse.

In the present invention, the raising animal immune ability product can be the drug for improving animal immune ability.

It is demonstrated experimentally that the amalgamation and expression VASP molecules of the present invention have the effect that：Promote animal lymph cell, red blood cell With the increase of leucocyte, 10% or more can be improved using animal body endolymph cell, red blood cell and the leucocyte content of VASP；It is bright The aobvious growth for inhibiting pathogenic microorganisms, can reduce using the amount of the pathogenic microorganisms of VASP up to 20% or more；Promote non-specific The secretion of antibody (IgG, IgG1, IgG2a) and disease specific antibody, using the content of non-specific antibody in the animal body of VASP 40%-60% can be improved；Promote the expression of gene involved in immunity, and then improve the immune anti-infection ability of animal, using VASP Animal attack malicious survival rate and significantly improve at least 60%, 10% or more weight gain.

The present invention is merged by genetic recombination, and building new has antibacterium, virus, fungi and parasite and immunological regulation The fusion antibacterial peptide gene of equal broad immunes anti-infection activity, expressed fusion protein VASP are further merged by MIC method researchs The immunological regulation of albumen VASP and its anti-infection activity, to obtain, broad-spectrum high efficacy is antimicrobial and the strong recombination of immunoregulation effect Albumen.

Description of the drawings

Fig. 1 is the digestion of target gene and PCR results in pVASP and transformant.

Fig. 2 is to carry pVASP plasmid agarose gel electrophoresis verification results greatly.

Fig. 3 is pVASP eukaryotic expression RT-PCR electrophoresis patterns.

Fig. 4, which is Validation in vitro pVASP supernatants, stimulates pig lymphocyte proliferation experiment result.

Fig. 5 is that fusion antibacterial peptide gene expresses supernatant to Escherichia coli, salmonella, staphylococcus aureus, hammer Bacterium growth inhibitory effect.

Fig. 6 is that peripheral white blood cells sum changes after experiment mice injects pVASP.

Fig. 7 be experiment mice inject pVASP after peripheral blood CD4+ CD8+T total number of cells grade ratios variation.

Fig. 8 is that experiment mice injects peripheral blood serum IgG after pVASP, IgG1, the variation of IgG2a levels.

Fig. 9 is that experiment mice injects changes of weight after pVASP.

Figure 10 is that TLRs gene expression doses change after experiment mice injects pVASP.

Figure 11 is that interleukin gene expression changes after experiment mice injects pVASP.

Figure 12 is that IFN-γ gene expression dose changes after experiment mice injects pVASP.

Figure 13 is that bacterium attacks malicious survival rate after experiment mice injects pVASP.

Specific implementation mode

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 present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

PVAX1 carriers in following embodiments are Invitrogen, Life technologies Products, product mesh Record number is Catalog No.V260-20.

Escherichia coli standard bacteria (G in following embodiments^-) it is ATCC (America Type Culture Collection) product, catalog number 25922；Salmonella (G^-, ATCC14028), America Type Culture Collection products, catalog number (Cat.No.) 14028；Staphylococcus aureus standard bacteria (G⁺, ATCC29213) and it is ATCC (America Type Culture Collection) product, catalog number 29213.Streptococcus standard bacteria (G⁺, ATCC19615), be ATCC (America Type Culture Collection) product, catalog number 19615.

Escherichia coli drug-fast bacteria (G-) in following embodiments and staphylococcus aureus resistance bacterium (G⁺) it is Sichuan University Field of Animal Epidemic Disease Control is provided with food security Key Laboratory of Sichuan Province, and the public can obtain the biomaterial from applicant, should Biomaterial is only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and is used.

Female KM mouse in following embodiments is People's Hospital, Sichuan Prov.'s institute of lab animals's product.

Embodiment 1, pig antibacterial peptide fusion protein (VASP) have bacteriostasis

The pig antibacterial peptide fusion protein that the present invention uses, entitled VASP carry the amino acid sequence of TPA secreting signal peptides Row are as shown in sequence 1 in sequence table, and the nucleotide sequence of the gene VASP of coding albumen VASP is as shown in sequence 2.

Wherein, sequence 1 1-25 be TPA secreting signal peptides, 26-160 be pig fusion antibacterial peptide, 161-166 is Tag molecule.Sequence 2 1-75 be TPA secretion signals DNA encoding peptide, 76-480 be pig fusion antibacterial peptide coding base Cause, 481-498 are code tag molecular gene.

One, the preparation of eukaryotic expression vector pVAX1-SP

DNA fragmentation between Hind III and Xbal the identification sequence of pVAX1 carriers is replaced with and carries TPA shown in sequence 2 The gene VASP of secreting signal peptide, keeps the other sequences of carrier constant, obtains recombinant vector, which is named as PVAX1-SP (abbreviation pVA-SP), carrier for expression of eukaryon pVA-SP can pig antibacterial peptide fusion proteins shown in expressed sequence 1 (VASP)。

Hind III and Xbal double digestions detection recombinant vector pVAX1-SP.

The results are shown in Figure 1, and A is recombinant plasmid pVAX1-SP electrophoresis patterns (1% Ago-Gel)；Lane M: DL5000Marker；Lane 1:PVAX1-SP, size 3385bp；Lane2:PVAX1 empty carriers, size 2999bp；B is Recombinant plasmid pVAX1-SP double digestions electrophoresis pattern (1% Ago-Gel)；Lane M:DL5000Marker；Lane1:pVAX1 2900bp and 100bp are obtained by Hind III, I double digestions of Xbal；Lane2:PVAX1 empty carriers, 3000bp；Lane3:Recombinate matter Grain pVAX1-SP obtains 2900bp and 500bp by Hind III, I double digestions of Xbal；It can be seen that VASP channel genes successfully PVAX1 carrier for expression of eukaryon, it was demonstrated that the eukaryotic expression recombinant plasmid for merging antibacterial peptide is built successfully.

Two, fusion recombinant plasmid pVASP eukaryotic cell expressions and In vitro biological activity detection

1, a large amount of extractions of recombinant plasmid

The carrier for expression of eukaryon pVASP that above-mentioned one obtains is converted to Escherichia coli Mach1-T1Phage Resistant (it is purchased from TransGene companies) in competent cell；Recombinant plasmid pVASP is largely extracted using conventional alkaline lysis.

(1) E.coli with recombination eucaryon plasmid pVASP is inoculated in 3ml LB/Kana antibiotic culture solutions, 37 DEG C shake culture is stayed overnight.The bacterium solution that 2ml is incubated overnight is taken, is inoculated into 200ml LB/Kana antibiotic culture solutions, 37 DEG C of concussions Cultivate 12-16h.

(2) it shifts in bacterium solution to 100ml centrifuge tubes, 4000rpm centrifuges 10min at 4 DEG C, removes supernatant as possible.Repeat this Operation is primary.

(3) bacterium solution precipitation is resuspended in 6ml Solution I, acutely shakes, thalline is made to be uniformly dispersed.

(4) 6ml Solution II are added, centrifuge tube is mildly overturned for several times to being uniformly mixed, centrifuge tube is placed in ice Upper 5min.

(5) the Solution III of 5ml precoolings are added, centrifuge tube is mildly overturned for several times to being uniformly mixed, is put on ice 10min is set, 10000rpm centrifuges 10min at 4 DEG C.

(6) supernatant is poured into 50ml centrifuge tubes, 12ml isopropanols, -20 DEG C of placement 10min, at 4 DEG C is added 10000rpm centrifuges 10min, removes most supernatant.

(7) precipitation is dissolved in the TE solution of 850ul, then transfers the solution into 1.5ml EP pipes, is added 400ul10mol/L NH4Ac overturn mixing for several times, place 10min on ice, and 10000rpm centrifuges 10min at 4 DEG C.

(8) supernatant is divided equally into (respectively about 620ul) in two 1.5ml EP pipes, it is mixed that 600ul isopropanols is then respectively added Even, -20 DEG C of placement 10min, 12000rpm centrifuges 10min at 4 DEG C, removes most supernatant.

100ul TE (A of RNase containing 40ug/ml) are respectively added in (9) two 1.5ml EP pipes, then dissolving precipitation will Solution is merged into a 1.5ml EP pipe, and 37 DEG C of water-bath 20min are with degradation of rna molecule.

(10) the 15%PEG8000+1.6mol/L NaCl of 200ul are added, overturns mixing for several times, places on ice 20min, then 14000rpm centrifuges 15min at 4 DEG C, removes most supernatant.

(11) precipitation is dissolved completely in 200ul TE, 80ul 10mol/L NH4Ac mixings is added, 280ul is then added Isopropanol overturns mixing for several times, places 10min on ice, and 12000rpm centrifuges 10min at 4 DEG C, removes most supernatant.

(12) 70% ethyl alcohol of 700ul washing precipitation and EP inside pipe walls is added, sucks 70% ethyl alcohol, carries out of short duration centrifugation, so It sucks remaining ethyl alcohol as possible afterwards, centrifuge tube is uncapped and is placed at room temperature for 10min, the ethyl alcohol of precipitation surface is made to volatilize.It is eventually adding 300-700ul TE dissolving precipitations.

The plasmid of extraction is subjected to size verification (see Fig. 2, Lane M with 0.7% agarose gel electrophoresis: DL5000Marker；Lane 1:PVAX1 zero load Lane 2-3:Recombinant plasmid pVA-SP).

2, the molecule packaging of pVASP eukaryotic expression recombinant plasmids

1) pVASP molecules are packaged to be the chitosan nano particle of pVASP

Chitosan carries out molecule packaging to carrier for expression of eukaryon pVASP：With ionic cross-linking (Bodmeier et al., 1989) chitosan (Chitosan, CS, 90% or more deacetylation, molecular weight are used：100kDa, Sigama company, USA, catalogue Number：900342) plasmid pVASP is wrapped up, i.e.,：The pVASP that chitosan aqueous solution (pH 5.5,5mg/ml) and above-mentioned 1 are obtained is molten Liquid (the solution for being mixed to get pVASP, tripolyphosphate and water, wherein a concentration of 0.5mg/ml of pVASP, tripolyphosphate Mass percentage be 0.1%) (mass ratio of chitosan and pVASP are 20:1) 65 DEG C of constant temperature water bath 5-10min, then Even mixing plasmid solution and chitosan solution 5-10min obtain the chitosan nano particle sample liquid of pVASP, use CNP-VASP To indicate.

Chitosan is subjected to molecule packaging as a control group to unloaded pVAX1 according to the method described above, the shell for obtaining pVAX1 is poly- Sugared nano particle.

2) the chitosan nano particle detection of pVASP

The chitosan nano particle sample liquid of the above-mentioned pVASP 1) obtained is concentrated 10 on a rotary evaporator in 45 DEG C Times, the volume of the concentrated liquid being collected into is the 1/10 of original volume, and supplement 0.9%NaCl physiological saline, which is softly beaten, is settled to 500 μ g/ Ml plasmid concentrations, in case cell transfecting and zoopery injection.

It is detected by 0.7% agarose gel electrophoresis, the nano particle of package is arrested in loading wells, fails to migrate Go out sample well, illustrates that recombinant plasmid is fully wrapped up by chitosan molecule, form effective nano particle.

3, the detection of nano particle transfected HEK 293 and target gene

When the HEK293 in culture plate it is adherent to 75% or so when, you can carry out cell transfecting, per hole be added contain 4 μ g The sample liquid of the chitosan nano particle of pVASP, while with TransLipid Transfection Reagent (Liperfectamine, Beijing Quan Shi King Companies, catalog number (Cat.No.)：FT101) 10ul mixes 4 μ g pVASP or pVAX1 plasmids, side Side mixing (obtaining Liperfectamine-VASP or Liperfectamine-pVAX1) is added dropwise；It is mixed and is dripped using preceding half an hour It is put into cell incubator after the completion of adding, 37 DEG C of 5%CO2 are incubated, and culture medium is replaced after 5 hours.After transfection for 24 hours, 48h, 72h take pictures to cell, and do RT-PCR detections；Collect CNP-VASP transfection HEK293 after for 24 hours, on the cell of 48h, 72h Clear liquid is spare.

HEK293 cell transfecting typesettings are shown in Table 1.

RT-PCR detection method is as follows：HEK293 cell total rnas after extraction transfection identify (P1 through RT-PCR：5- GTTTAAACTTAAGCTTATGGATGCAATG-3；P2：5-AAACGGGCCCTCTAGACTAATGGTGATG-3).

The results are shown in Figure 3, Lane 1：Liperfectamine-VASP；Lane 2：Liperfectamine-pVAX1； Lane3,4,5：VASP–CS；M:DNA marker；As can be seen that Liperfectamine-VASP and VASP-CS transfections There is specific amplified purpose band, about 125bp in HEK293 cells.And there is not feature in Liperfectamine-pVAX1RT-PCR Band.

The above results show no matter packed by which kind of material, and pVASP recombinant plasmids can express in HEK293 cells The corresponding mRNA of target gene fragment.

By PCR product and quantitative analysis destination gene expression, transfection 48H expression quantity highests are found.

Table 1.HEK293 transfects typesetting

4, pVASP is on lymphopoietic influence

1) preparation of pig lymphoblast is hidden

Pig vena cava anterior peripheral blood 5ml is taken with anticoagulant tube (EDTA-2K anti-freezings) under aseptic condition, according to lymphocyte point Pig lymphocyte is hidden in the separation of chaotropic specification.The lymphocyte being separated to is diluted to final concentration of cells with 1640 complete culture solutions 2×10⁶/ ml divides into the Tissue Culture Dish of a diameter of 10cm, per ware 10ml, finally adds final concentration of 10 μ g/ml's CoA (con A, the Shanghai bio tech ltd Heng Yuan, No. CAS：11028-71-0) carry out culture stimulation, 5% CO₂, cultivate for 24 hours in 37 DEG C of cell incubators.

2) the biological activity detection of novel antimicrobial peptide track fusion product

By the research of front, transfection is found 48 hours through TransLipid Transfection Reagent transfections The expression quantity highest of foreign gene, therefore lymphocyte stimulation experiment expressed supernatant using 24,48 hours HEK293 cell transfectings.

It is to swash through CoA that wherein control wells C Negative, which use the lymphocyte without CoA stimulation activation, C Positive, Lymphocyte living observes lymphocyte activity.

Lymphocyte stimulation experiment is specific as follows：

1) when for 24 hours, the cell in culture dish is collected, 2500rpm centrifuges 5min and collects cell；

2) cell is cleaned 2 times with PBS or 1640 complete mediums, each 2500rpm centrifuges 5min；

3) cell is washed 1 time with the 1640 culture mediums of-MM of α containing 20mg/ml, 1500rpm centrifuges 15min；

4) cell is adjusted to about 6x10 with the 1640 culture mediums of-MM of α containing 20mg/ml⁶/ml；

What 96 orifice plate experimental ports addition, 50 μ l target cells (lymphoblast activated through CoA) and 50 μ l above-mentioned 3 were obtained CNP-VASP transfect HEK293 after for 24 hours, the cell supernatant of 48h and 72h.Equipped with 1640 complete mediums of RPMI, phosphoric acid buffer Liquid PBS, lymphocyte blank control, pVAX1 chitosan nano particle transfection HEK293 after for 24 hours, on the cell of 48h and 72h Clear liquid compares, and each sample sets 2 repeating holes, is placed in cell incubator culture 5%CO2,37 DEG C of 48h.

5) when 48h, 96 orifice plates are taken out, 10 μ l of CCK8 are added per hole and continue cell incubator culture 5%CO2,37 DEG C of 2h.

6) 96 orifice plates are taken out, are detected per hole OD450 with Bio-Reader3550.

As a result as (VASP is pVASP groups to Fig. 4, is transfected after HEK293 for 24 hours, on the cell of 48h and 72h for CNP-VASP Clear liquid；The chitosan nano particle that pVAX1 groups are pVAX1 transfect after HEK293 for 24 hours, the cell supernatant of 48h and 72h), can be with Find out, pVASP groups supernatant relatively control pVAX1 groups proliferation becomes apparent from, this illustrates equal after fusion protein VASP is wrapped up with two kinds of materials Energy expression has biological activity, and pig lymphoblast can be stimulated to be proliferated, and its expression product of transfection 48h is possible at most, shows Write stimulation lymphopoiesis (P<0.05).

5, pVASP expresses the detection of supernatant bacteriostatic activity and minimal inhibitory concentration MIC relative methods

Pig antibacterial peptide fusion protein is measured to Escherichia coli standard bacteria (G^-), salmonella, staphylococcus aureus standard Bacterium (G⁺) (hereinafter referred to as S-G⁺), streptococcus antibacterial situation, the specific method is as follows：

4 kinds of bacterium bacterial strain inoculations are activated and cultivate it first and are in exponential phase of growth (OD₆₀₀About 0.5), it then uses LB culture solutions are diluted to OD₆₀₀About 0.005, the bacterium solution after dilution is inoculated into 96 porocyte culture plates, 100 μ L/ are per hole, often A kind of a bacterium of 96 porocyte culture plates.

It is handled in the following way containing germy 96 porocyte culture plates for each：By 100 μ LCNP-VASP transfections Gently mixing, each sample set 3 repeating holes in the cell supernatant addition experimental port of 48h after HEK293.

After 96 porocyte culture plates are set 37 DEG C of incubators incubation 16h, microplate reader (Bio-Reader 680) is used to examine respectively It surveys per hole OD₆₀₀Value.

S-G^-And S-G⁺Four kinds of antibiotic gradients it is as shown in table 2,

Table 2, reference culture antibiotic gradient

Note：In table 2, " --- " indicates to be free of kanamycins.

Its bacteriostatic activity is detected by the following method：4 kinds of bacterium bacterial strain inoculations are activated and cultivate it first and are in index Growth period (OD₆₀₀About 0.5), then OD is diluted to LB culture solutions₆₀₀About 0.005, the bacterium solution after dilution is inoculated into 96 In porocyte culture plates, 100 μ L/ are per hole, a kind of each bacterium of 96 porocyte culture plates.

It is handled in the following way containing germy 96 porocyte culture plates for each：100 μ L sample liquid are added real Gently mixing, each sample set 3 repeating holes in verifying；

The culture supernatant of 48h after the CNP-VASP transfected HEK 293s that wherein sample liquid obtains for the step 3 of embodiment 1 The culture supernatant (VAX1) of 48h after the chitosan nano particle transfected HEK 293 of liquid (L-VAPA) or pVAX1.

Each bacterium is all provided with 4 kinds of antibiotic gradients as positive control (antibiotic and LB culture solutions)；Each bacterium, which is all provided with, only to be added LB culture solutions set not bacteria-containing LB culture solutions as blank control as negative control.

After 96 porocyte culture plates are set 37 DEG C of incubators incubation 16h, microplate reader (Bio-Reader3350) is used to examine respectively It surveys per hole OD₆₀₀Value measures the antibacterial activity of fusion antibacterial peptide.

As a result such as Fig. 5, it can be seen that compared with VAX1, the different diluent of L-VASP is to Staphylococcus aureus and hammer Bacterium all has apparent inhibiting effect (P<0.05)；(P also more significant to the inhibiting effect of G- bacterium<0.05), 48H supernatants dilute Still there is significant bacteriostatic activity at 32 times, is approximately close to 50 μ g/ml ammonia benzyl antibiotic or 100 μ g/ml kanamycins.Above-mentioned knot Fruit shows that New Fusion antibacterial peptide VASP has significant bacteriostatic activity, lays the foundation for subsequent experimental, selects Staphylococcus aureus Bacterium and Escherichia coli are as follow-up animal challenge test bacterial strain.

Embodiment 2, fusion eukaryotic expression recombinant plasmid biological activity research in Mice Body

1, endotoxic detection

The endotoxin content of plasmid, method reference are detected with " gel method "《Reagents specification》, the specific method is as follows：

(1) prepared by positive control sample：Endotoxin standard (the Shanghai bio tech ltd Ha Ling, 150600) is taken, With the solution for standby that inspection water compound concentration is 2 times of sensitivity λ=0.25Eu/ml；

(2) negative control：Inspection water；

(3) sample is detected：Effective extension rate (the MVD=cL/ of calculator must be sought according to detection sample endotoxin limit value λ), sample is diluted spare；

(4) reagents, breaking off ampoule neck is taken simultaneously to mark, wherein 1 flag positive control pipe, 1 flag negative control pipe, 2 Make inspection quality control, 1 flag examines product positive control pipe, totally 5；

(5) be added in negative control pipe, positive control pipe and inspection quality control respectively as requested 0.2ml inspections water, 0.1ml checks (real with endotoxin solution and 0.1ml the inspection water+0.1ml detection samples of a concentration of 2 times of λ of water+0.1ml Apply the chitosan nano particle sample liquid of the pVASP of the acquisition of example 1 or the chitosan nano particle sample liquid of pVAX1), gently shake up It is to be checked；

(6) previous step is waited for that sample is vertically put into thermostat, 37 DEG C are incubated 60 minutes, read result.

As a result it has no that sample cell has gel-forming, shows the chitosan nano particle sample of eucaryon plasmid pVASP and pVAX1 Liquid does not contain endotoxin.

2, experiment mice is grouped and is immunized

1) it is grouped

20 28 small female mices in age in days Kunming, weight about 25-30g, random 2 groups of grouping, every group of 10 mouse are divided into experimental group And control group.

2) it is immunized

Experimental group VASP:For by the obtained CNP-VASP intraperitoneal injection of mice of embodiment 1, injection dosage is every mouse The chitosan nano particle of 100 μ g pVASP.

Control group pVAX1:Chitosan nano particle sample liquid intraperitoneal injection of mice for the pVAX1 for obtaining embodiment 1, Injection dosage is the chitosan nano particle of every 100 μ g pVAX1 of mouse.

3, mouse inoculation and drug-fast bacteria attack poison

The experimental group VASP and control group pVAX1 mice immunisations obtained above-mentioned 2 selects high drug resistance golden yellow after 28 days Color staphylococcus and Escherichia coli are incubated overnight after being inoculated with LB culture mediums, and detection OD values directly use 0.2ml 1.0 or so Original bacteria liquid injects mouse peritoneal.To 10 mouse in every group, the different drug-fast bacterias of each point of 5 injections are observed mouse invasion rate and are deposited Motility rate.

Respectively before gavage, gavage the 7th day, gavage the 14th day, gavage the 21st day and gavage take every mouse within the 28th day Tail vein carries out following immunology detection：After 30 μ L whole bloods and 30 μ L physiological saline mixings blood routine is done in blood counting instrument Analysis；50 μ L whole bloods do Flow cytometry；Before mouse immune and 3 weeks after immune, poison latter week is attacked from tail vein blood, separation Serum, MBP enzyme linked immuno-adsorbent assay technology (ELISA) detect IgG, IgG1 and IgG2 changes of contents in serum.Before and after immune animal The set time weighs the weight of mouse weekly respectively, and makes a record, and can substantially find out VAP to mouse from average weight variation The influence of growth；100 μ L whole bloods extract real-time quantitative gene involved in immunity after RNA, and quantitative relevant primer is shown in Table 3.

Table 3 quantifies primer

Every group of mouse peripheral blood immunocyte variation is as shown in fig. 6, immune rear 0d-28d, experimental group VASP and control group The quantity of leucocyte of pVAX1 mouse obviously increases (P<0.05).CS chitosans wrap up pVASP nano particle experimental mices periphery Blood leukocytes quantity is all remarkably higher than control group, illustrates that CS wraps up pVASP nano particle energy effective stimulus immune cell propagations, number Amount improves.

Fig. 7 shows that CD4+T the and CD8+T cell quantities of experimental mice significantly increase (P after 14 days compared with control group< 0.05), and CD4/CD8 ratios increase.The universal CD8+T ratios of mouse are high before experiment, after inoculation CD4+T quantity and ratio it is continuous on It rises, display mouse is obviously reinforced to the apparent response of cellular immunity.

Fig. 8 shows IgG, IgG1 and IgG2 levels in experimental mice serum after inoculation respectively at 21-28 days, 7 days and 14- Obviously rise (P compared with control mice within 28 days<0.05), illustrate that the humoral immunity of mouse is also obviously reinforced.

Fig. 9 shows that weight increases compared with control mice experimental mice weight after inoculation, and the difference in later stage reaches notable Horizontal (P < 0.05)；Illustrate to be inoculated with the growth-weight gain that pVASP effectively promotes mouse.

Shown in Figure 10, the expression of experimental mice TLR1, TLR4, TLR6 and TLR9 gene 21-28 days after inoculation It was significantly improved (P < 0.05) compared with control mice with 7-28 days；Show that immunized mice more rapidly can efficiently identify cause of disease Microorganism, inherent immunity ability get a promotion.

Shown in Figure 11, IL-1 principal biologicals function carries out immunological regulation in local low concentration, and collaboration stimulation APC and T is thin Born of the same parents activate, and promote B cell proliferation and secretory antibody.Experimental result shows that pVASP inoculations can be obviously improved IL-1 points of mouse Bleeding puts down (P<0.05).Equally, IL-2, IL-4, IL-7 and IL-15 transcriptional level of experiment mice also different degrees of rising, Illustrate that expression can effectively facilitate the expression of cytokine profiles in New Fusion antibacterial peptide VASP genosomes, and it is thin to reinforce animal immune The activation of born of the same parents and Immunity regulation.

Shown in Figure 12, experimental mice blood IFN-γ transcriptional expression is significantly higher than control group (P for 14-28 days<0.05), exist IFN-γ reaches peak at 21 days.The rising of IFN-γ illustrates that New Fusion antibacterial peptide VASP genes promote Th1 type immune responses.

Shown in Figure 13, challenge viral dosage uses two plant height drug-resistant bacterias：G+ bacterium：Staphylococcus aureus, G- bacterium：Escherichia coli It carries out after attacking poison, is spaced after intraperitoneal injection 6 hours, 12 hours, 24 hours to 5 days and observes survival rates and lesion, death condition.Knot Fruit shows that New Fusion antibacterial peptide VASP genes can be effectively protected Mice Inoculated, and inoculation experiments group is to Staphylococcus aureus The resistance that bacterium, challenge with E.coli infect significantly is reinforced, the experimental group death rate 0/5 (G+) -0/5 (G-), and blank control group Dead 4/5.

Sequence table

<110>Sichuan University of Hai Jinzhuo Bioisystech Co., Ltd before Shenzhen

<120>Merge the preparation and application of antibacterial peptide VASP biological agents in Novel pig source

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 166

<212> PRT

<213>Artificial sequence

<400> 1

Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

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

20 25 30

Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro Phe

35 40 45

Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro Arg

50 55 60

Phe Pro Pro Arg Phe Pro Gly Lys Arg Gly Ser Gly Asp Asp Asp Asp

65 70 75 80

Lys Val Arg Arg Phe Pro Trp Trp Trp Pro Phe Leu Arg Arg Gly Ser

85 90 95

Gly Asp Asp Asp Asp Lys Arg Ile Ile Asp Leu Leu Trp Arg Val Arg

100 105 110

Arg Pro Gln Lys Pro Lys Phe Val Thr Val Trp Val Arg Gly Ser Gly

115 120 125

Asp Asp Asp Asp Lys Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg

130 135 140

Phe Cys Val Cys Val Gly Arg Gly Gly Ser Gly Asp Asp Asp Asp Lys

145 150 155 160

His His His His His His

165

<210> 2

<211> 498

<212> DNA

<213>Artificial sequence

<400> 2

atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgcccagcg gtacccctac cggatccaga tctaggagac gtccccgacc cccatatttg 120

ccaaggccaa ggccacctcc gtttttccca ccaaggttgc caccacgtat cccaccaggg 180

ttcccaccaa ggttcccacc acggttcccc ggaaaacggg gatccggaga tgacgatgac 240

aaggtacgac gtttcccatg gtggtggcct ttcttgcgac gtggatccgg agatgacgat 300

gacaagagga ttattgactt gttgtggaga gtacgtcggc cacagaaacc caaatttgtg 360

actgtatggg tcagaggatc cggagatgac gatgacaaga ggggaggtcg cctgtgctat 420

tgtaggcgta ggttctgcgt ctgtgtcgga cgaggaggat ccggagatga cgatgacaag 480

catcatcacc atcaccat 498

Claims (10)

1. Be following a)-e 1. a kind of protein) in any protein：

   A) amino acid sequence includes the protein of amino acid sequence shown in sequence 1 in sequence table；

   B) amino acid sequence amino acid residue shown in sequence in sequence table 1 forms；

   C) by amino acid sequence defined by a) or b) by the substitution of one or several amino acid residues and/or missing and/or Addition and the protein with raising animal immune ability function；

   D) amino acid sequence defined by and a) or b) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology and the protein with raising animal immune ability function；

   E) a)-d) in it is any defined by protein N-terminal and/or C-terminal connection label after obtained fusion protein.
2. 2. encoding the nucleic acid molecules of albumen described in claim 1.
3. 3. nucleic acid molecules according to claim 2, it is characterised in that：The nucleic acid molecules are following 1) -4) in it is any Shown in nucleic acid molecules：

   1) its coded sequence includes sequence 2 in sequence table；

   2) its coded sequence is sequence 2 in sequence table；

   1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of albumen described in claim 1；

   1) or 2) 4) with the DNA molecular that limits with described in 80% or more or 90% or more homology and coding claim 1 The DNA molecular of albumen.
4. 4. following 1) -4) any one of biomaterial：

   1) expression cassette containing nucleic acid molecules described in Claims 2 or 3；

   2) recombinant vector containing nucleic acid molecules described in Claims 2 or 3；

   3) recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3 or transgenic cell line；

   4) contain the nano particle of the recombinant vector.
5. 5. nano particle according to claim 4, it is characterised in that：The nano particle is by the recombinant vector shell The nano particle that glycan or cationic-liposome wrap up.
6. 6. the biomaterial described in nucleic acid molecules or claim 4 described in protein or Claims 2 or 3 described in claim 1 Application in following C1 or C2 or C3：

   C1, animal immune ability is being improved；

   C2, it prepares and improves animal immune ability product；

   C3, preparation act on the anti-infective product of wide spectrum of animal.
7. 7. applying according to claim 6, it is characterised in that：The raising animal immune ability be following M1-M5 in extremely Few one kind：

   M1, the growth for inhibiting pathogenic microorganisms；

   M2, the increase for promoting immunocyte；

   M3, promote vaccine-induced immune response；

   M4, promote cellular immunity and/or humoral immunity；

   M5, animal development and growth-weight gain are improved；

   And/or the pathogenic microorganisms is specially Escherichia coli, salmonella, staphylococcus aureus and streptococcus, it is described to exempt from Epidemic disease cell is specially lymphocyte or leucocyte；

   And/or the antibody specific is IgG, IgG1 and/or IgG2a；

   And/or the wide spectrum it is anti-infective for antibacterium, antiviral, true antibacterial or anti parasitic.
8. 8. any products of following X1 or X2：

   X1, biological agent contain following X3a, X3b or X3c：

   Any protein in X3a, claim 1-3；

   X3b, claim 4 or 5 nucleic acid molecules；

   Biomaterial described in X3c, claim 6；

   X2, the reagent set for improving animal immune ability, by above-mentioned X1 and antibiotic group at.
9. 9. a kind of method improving animal immune ability, including protein or claim 2 described in claim 1 are applied to animal Or 3 biomaterial described in the nucleic acid molecules or claim 4 or the biological agent or the reagent set, described in raising The immunocompetence of animal.
10. 10. applied described according to claim 6 or 7 or product according to any one of claims 8 or claim 9 described in method, It is characterized in that：The animal is H1 or H2：

    H1, mammal；

    H2, mouse.