CN101157724A - Modified recombinant porcine alpha interferon protein and coding gene and uses thereof - Google Patents

Abstract

The present invention discloses a recombinant protein, an encoding gene and the application thereof. The recombinant protein is the protein of (a) or (b) as follows: (a) the protein which is composed of an amino acid sequence which is show by a sequence 1 in a sequence table; (b) the protein which is derived from (a) by replacement and/or deletion and/or addition of one or a plurality of amino acid residues of the amino acid sequence of the sequence 1 in the sequence table and has the activities of interferon and thymosin. The recombinant protein of the present invention can be expressed high effectively in the escherichia coil, the expression amount can achieve more than 60 percent of the total protein of the bacteria, the renaturation recovery rate can achieve more than 70 percent, which is much higher than the renaturation efficiency of the existing interferons, furthermore, the present invention has short cycle and low cost.

Description

A kind of improvement recombinant porcine alpha interferon protein and encoding gene and application

Technical field

The present invention relates to a kind of reorganization improvement porcine alpha interferon protein and encoding gene and application.

Background technology

Interferon, rabbit is that cell induces action-reaction and produces the protein molecular of the natural generation of a class of justacrine virus infection or the various synthetic biology that reaches, and molecular weight is 15,000-21,000 dalton.The interference of Que Dinging have two big classes: I type and II type.I type Interferon, rabbit comprises IFN-α, IFN-β, IFN-ω and four hypotypes of IFN-τ, and II type Interferon, rabbit mainly contains IFN-γ.The gene order of pig interferon α, β, γ all reports, and realizes recombinant expressedly in protokaryon and eukaryotic expression system, and expression product has antiviral activity, for the large-scale production and the application of genetic engineering interferon provides foundation.

People such as nineteen sixty-five Goldestein have been separated to bioactive polypeptide quasi-molecule, called after thymosin (Thymosin) from animal thymus.Goldestein in 1972 etc. are further purified the component of resulting molecular weight at 1-15KD, and (Thymosin, TF5), Zadaxin component 5 is used for zooscopy and clinical observation, and the low effect of compensation thymus function is arranged to be referred to as Zadaxin component 5.Thymosin alpha 1 (Thymosin α 1, T α 1) is one of the strongest active polypeptide in Zadaxin component 5 polypeptide mixture, has the function that promotes the differentiation of T-lymphocyte, maturation and strengthen cellular immunization.Its peptide sintetics has been applied to clinically at present abroad, has been used for research such as the treatment of tumour, hepatitis B, third liver and immune deficiency etc., and has obtained good effect.

Summary of the invention

The purpose of this invention is to provide a kind of reorganization improvement porcine alpha interferon protein.

Recombinant protein provided by the present invention, name is called PoIFN-α, has the biological activity of Interferon, rabbit and Zadaxin, is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b), and has Interferon, rabbit and Zadaxin is active by (a) deutero-protein with replacement and/or disappearance and/or the interpolation of the aminoacid sequence of sequence in the sequence table 1 through one or several amino-acid residue.

In order to make the PoIFN-α in (a) be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label

Residue

Sequence

Poly-Arg	5-6 (being generally 5)	RRRRR
			Poly-His	2-10 (being generally 6)	HHHHHH
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

Above-mentioned (b) but in PoIFN-α synthetic, also can synthesize its encoding gene earlier, carry out biology according to following method again and express and to obtain.The encoding gene of PoIFN-α in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna of sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or at the encoding sequence of its 5 ' end attach signal peptide, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

For the ease of proteic secreting, expressing, signal peptide sequence on also can adding at the N-terminal of described PoIFN-α.

The encoding gene of above-mentioned recombinant protein (PoIFN-α) also belongs to protection scope of the present invention.

Described recombinant protein gene specifically can be following 1) or 2) gene:

1) its nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table;

2) can be with 1 under stringent condition) the dna sequence dna hybridization that limits and the dna molecular of the described recombinant protein of encoding.

Sequence 2 in the sequence table is made up of 615 deoxyribonucleotides, and deoxyribonucleotide is an encoding sequence from 5 ' end 1-612 position, and coding has the dna molecular of the amino acid residue sequence of sequence 1 in the sequence table.

Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, 65 ℃ of hybridization and wash film down with this solution.

The recombinant expression vector, transgenic cell line and the reorganization bacterium that contain above-mentioned recombinant protein gene also belong to protection scope of the present invention.

Another object of the present invention provides a kind of method of express recombinant protein.

The method of express recombinant protein provided by the present invention is that described recombinant protein gene is imported host cell by expression vector, and screening obtains the reconstitution cell of express recombinant protein, cultivates this reconstitution cell, expresses obtaining recombinant protein.

Described expression vector is pBV220, pET carrier series, pQE carrier series or pPIC9K, is specially pBV220.

Described host cell is colibacillus DH5 α, TB1 or BL21 (DE3), is specially colibacillus DH5 α.

Described expression can be abduction delivering, and the abduction delivering temperature is specially 42 ℃, and the abduction delivering time is 6-8 hour.

The 3rd purpose of the present invention provides a kind of medicine that prevents and/or treats swine disease poison infectious diseases.

The medicine that prevents and/or treats swine disease poison infectious diseases provided by the present invention, its activeconstituents is above-mentioned recombinant protein.

Described swine disease poison infectious diseases comprises porcine reproductive and respiratory syndrome (blue otopathy), porcine epizootic diarrhea, transmissible gastroenteritis, rotavirus infection, swine fever, the infection of PCV-II II type, pseudoabies, porcine influenza, hydroa and parvovirus etc.

The described medicine that prevents and/or treats swine disease poison infectious diseases can be by injection, injection, collunarium, eye drip, infiltration, absorption, physics or chemistry mediation method importing body such as muscle, intracutaneous, subcutaneous, vein, mucosal tissue; Or mixed by other materials or wrap up the back and import body.

The described consumption that prevents and/or treats swine disease poison infectious disease medicament be generally 400,000 U/ heads/time, successive administration or administration every other day, general administration 3-7 days.

Recombinant protein of the present invention can efficiently express in intestinal bacteria, and expression amount reaches more than 60% of bacterial protein, and the recombinant protein renaturation rate of recovery reaches more than 70%, and considerably beyond the annealing efficiency of present Interferon, rabbit, and the cycle is short, and cost is low.Recombinant protein of the present invention has anti-infectious function to porcine reproductive and respiratory syndrome (blue otopathy), porcine epizootic diarrhea, transmissible gastroenteritis, rotavirus infection, swine fever, the infection of PCV-II II type, pseudoabies, porcine influenza, hydroa, parvovirus, can obviously improve animal immunizing power, increase resistance against diseases.

Description of drawings

Fig. 1 detects the pcr amplification result of recombinant protein gene for agarose gel electrophoresis

The attach most importance to splicing synoptic diagram of histone gene of Fig. 2

Fig. 3 detects recombinant protein in expression in escherichia coli product result for SDS-PAGE

Fig. 4 is recombinant protein matrix assisted laser desorption ionization flight time mass spectrum figure

Fig. 5 is the SDS-PAGE detected result of purified recombinant protein

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

All primers synthesize and examining order is finished by Beijing three rich polygala root biotech firms.

Synthetic and the expression of embodiment 1, recombinant protein gene

According to the merger of codon and intestinal bacteria skewed popularity to amino acid code, the present invention has designed the Thymosin alpha 1 encoding gene that contains intestinal bacteria hobby codon, and it is coupled to 5 of porcine alpha-IFN gene ' end, make up recombinant protein PoIFN-α gene, thereby improve this recombinant protein expression of gene level, the splicing synoptic diagram of PoIFN-α as shown in Figure 2.Concrete experimental technique and result are as follows:

One, the Thymosin alpha 1 gene is synthetic

With reference to Thymosin alpha 1 gene order (GENBANK number: J02524), again majorizing sequence, replace rare codon, regulate AT content, it can be efficiently expressed in intestinal bacteria, and at 5 of gene ' end introducing initiator codon and EcoRI restriction enzyme digestion sites, 3 ' end is introduced glycine/Serine catenation sequence and BamHI restriction enzyme digestion sites, transfer to Beijing three rich polygala root biotech firms and adopt the synthetic Thymosin alpha 1 gene order of optimizing of overlapping PCR method, its nucleotide sequence is shown in sequence in the sequence table 3.

Two, the amplification of porcine alpha-IFN gene

With reference to the porcine alpha-IFN gene sequence (GENBANK number: M28623), its mature protein coding sequence two ends design primer (upstream: 5 '-GGC AGATCTGGTGGCGGGGGAAGCTGTGACCTGCCTCAGACCC-3 ', the downstream: 5 '-CC GGATCCTTACTCCTTCTTCCTGAGTCTGTCT-3 '), and at 5 of gene ' end introducing glycine/Serine catenation sequence and BglII restriction enzyme digestion sites, 3 ' end is introduced the BamHI restriction enzyme digestion sites, and it is synthetic to transfer to Beijing three rich polygala root biotech firms.Extracting the pig liver genomic dna, is that template is carried out pcr amplification with the genomic dna, and the PCR reaction system contains template 1 μ g, each 50pmol/L of upstream and downstream primer, cumulative volume 50 μ L.Reaction conditions is 94 ℃, 8min; 94 ℃, 1min, 54 ℃, 1min, 72 ℃, 1min, 30 circulations; At last in 72 ℃, 10min.The PCR product detects through 1.2% agarose gel electrophoresis, and detected result as shown in Figure 1.Wherein, M is a dna molecular amount standard; 1 is PCR product electrophoresis result; 2 negative results of comparison.The result shows at the 520bp place band is arranged, reclaim this band with and pMD18-T carrier (TaKaRa company) is connected, transformed into escherichia coli DH5 α competent cell screens hickie, to the positive cloning and sequencing of PCR detected result.Sequencing result shows that the nucleotide sequence of the porcine alpha-IFN gene of pcr amplification is shown in sequence in the sequence table 4.

Three, the splicing of PoIFN-α gene

The Thymosin alpha 1 gene and the fragment behind the porcine alpha-IFN gene pcr amplification of above-mentioned acquisition are cut T with BamHI and BglII enzyme respectively ₄Dna ligase connects rear clone and goes into the pMD18-T carrier, transformed into escherichia coli DH5 α competent cell, and the screening hickie is to the positive cloning and sequencing of PCR detected result.The right-on clone of sequencing result is the recombinant protein gene, its nucleotide sequence shown in sequence in the sequence table 2, the PoIFN-α of encoding amino acid sequence shown in sequence in the sequence table 1.The recombinant vectors called after pMD18-PoIFN-α that will contain the PoIFN-α gene of nucleotide sequence shown in sequence in the sequence table 2.

Four, the purifying of recombinant protein expression of gene and expression product thereof

1, the structure that contains the intestinal bacteria recombinant expression vector PoIFN-α/pBV220 of recombinant protein gene

With the right-on plasmid pMD18-PoIFN-α that contains the recombinant protein gene of sequencing result be inserted into after with restriction enzyme BamHI and EcoRI double digestion the pBV220 prokaryotic expression carrier (contain the establishment and the application thereof of the protokaryon efficient expression vector of PRPL promotor. viral journal, 1990,6 (2): between BamHI 111～116) and the EcoRI restriction enzyme site, transformed into escherichia coli DH5 α competent cell, obtain positive reorganization bacterium, to detect with enzyme through PCR and cut recombinant vectors called after PoIFN-α/pBV220 that evaluation contains PoIFN-α, with the reorganization bacterium called after DH5 α-PoIFN-α/pBV220 that contains PoIFN-α/pBV220 of this recombinant vectors transformed into escherichia coli DH5 α competent cell acquisition.

Above-mentioned reorganization bacterium is inoculated in the LB substratum, and under 30 ℃ of conditions, shaking culture is to OD on 200 rev/mins of (rotation radius is 13mm) shaking tables ₆₀₀Value is 1, and a part is collected thalline, and another part continues to induce under 42 ℃ 6 hours.After cultivating end, 5000g collected thalline in centrifugal 10 minutes, to induce the thalline of back collection and carry out the SDS-PAGE electrophoresis detection without the thalline of inducing collection, detected result as shown in Figure 3, wherein, M is a protein molecular weight standard, and 1 is without inductive thalline SDS-PAGE electrophoresis result, and 2 for inducing the SDS-PAGE electrophoresis result of back thalline.Detected result shows, locates to occur a protein band (arrow shows) about 23kD, conforms to recombinant protein expection molecular weight.(MALDI-TOF MS) identifies by the matrix assisted laser desorption ionization flight time mass spectrum, the result as shown in Figure 4, the main peak and the pig interferon α that show recombinant protein peptide figure coincide, and prove recombinant protein gene (PoIFN-α) correctly expression in intestinal bacteria.Determine that through the shallow layer gel scanner expression product proportion in whole bacterial protein is 65%.

2, the fermentative production of recombinant protein

1) preparation of reorganization bacterium DH5 α-PoIFN-α/pBV220 seed bank

Recombinant expressed bacterium DH5 α-PoIFN-α/pBV220 is inoculated in 20ml by 1% volume ratio contains in the LB liquid nutrient medium of 100 mcg/ml Amp (penbritin) 30 ℃, 200rmp shaking culture 8-10h.Be warming up to 42 ℃ of inducing culture 4h, draw flat board, picking 5-10 single bacterium colony prepares seed bank: picking 5-10 single bacterium colony is cultured to OD in the LB liquid nutrient medium ₆₀₀Value is about 0.5, adds the mixed of 250ul 50% glycerine by 750ul bacterium liquid, and-20 ℃ of preservations are standby.

2) reorganization bacterium DH5 α-PoIFN-α/pBV220 fermentation seed liquid preparation

The reorganization bacterium DH5 α-PoIFN-α/pBV220 of-20 ℃ of preservations is inoculated in the 200ml2*YT nutrient solution (peptone 16 grams per liters, yeast powder 10, sodium-chlor 5 grams per liters) by the amount of 0.05% volume, and 30 ℃, 220rpm are cultivated 10-12h, make fermentation seed liquid.

3) reorganization bacterium DH5 α-PoIFN-α/pBV220 fermentative production

A, yeast culture stage

Fermention medium (grams per liter): peptone 5, yeast powder 5, KH ₂PO ₄2, K ₂HPO ₄4, Na ₂HPO ₄12H ₂O7, (NH ₄) ₂SO ₄1.2, NH ₄Cl 0.2, MnSO ₄5H ₂O 0.001, CoCl ₂6H ₂O 0.004, Na ₂MoO ₄2H ₂O0.002, ZnCl ₂0.002, CuSO ₄5H ₂O 0.001, H ₃BO ₄0.005, FeSO ₄7H ₂O 0.02, CaCl2H ₂O0.02, MgSO ₄7H ₂O 0.3, defoamer 0.2.This fermention medium water preparation.Behind the high-temperature sterilization, add the Amp of 1mL 100mg/ml in every liter of substratum, press the 5-10% volume and insert seed liquor, 35 ℃ of aeration-agitations were cultivated 4 hours, were cooled to 30 ℃ after 4 hours and continued cultivations.Along with the growth of thalline, the carbon source in the substratum consumes gradually in the culturing process, not regrowth of thalline after carbon source runs out of, and dissolved oxygen gos up (rising 30%), begins the flow feeding substratum this moment.Consisting of of this supplemented medium: glycerine 150ml/L, peptone 30g/L, yeast powder 30g/L, MgSO ₄7H ₂O 5.5mg/L, surplus is a water.Flow acceleration (is set DO=30% by dissolved oxygen (DO) control, when DO greater than 30%, open stream and add the pump fed-batch medium, DO progressively descends, when dropping to 30%, DO closes) with the dirty pump that adds, approximately per hour stream adds the 26-35ml substratum, keeps pH value 7.2 with 3M NaOH and 10% phosphoric acid in the culturing process.Dissolved oxygen maintains more than 30%, cultivates 2-3 hour, and prepares to induce for 30 ℃.

B, abduction delivering stage

Continue the flow feeding substratum, be warming up to 42 ℃ of abduction delivering target proteins, induce to stop after 7 hours cultivating.

3, the purifying of recombinant protein

Get the above-mentioned fermented liquid of 5L, 5000g collected thalline in centrifugal 10 minutes, through PBS damping fluid (NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/L, pH8.0) the washing back is resuspended, ultrasonic (Φ 10 probes, ultrasonic 7 seconds, interval 5 seconds) made the thalline broken wall in 30 minutes, under 4 ℃ of conditions, centrifugal 10 minutes collecting precipitations of 10000g, the washing precipitation of PBS damping fluid, use sex change damping fluid (6mol/L Guanidinium hydrochloride, 2mmol/L EDTA, 50mmol/L TrisHCl then, 10mmol/L DTT, pH8.5) dissolution precipitation, under 4 ℃ of conditions, 10000g collected supernatant in centrifugal 10 minutes, promptly obtain metaprotein solution, metaprotein solution is slowly joined renaturation buffer (0.5mol/L L-Arg; 2mmol/L EDTA, 20% glycerine; 0.9mmol/L GSSG, 0.1mol/L TrisHCl, pH8.0) in, 4 ℃ left standstill 48 hours, under 4 ℃ of conditions, centrifugal 20 minutes of 10000g collects supernatant, is the solution that contains recombinant protein.

Adopt the Bradford method to measure the protein concentration of recombinant protein solution, calculate the renaturation rate of recovery.Calculation formula is: recombinant protein strength of solution * volume/metaprotein quality, the result shows that the renaturation rate of recovery of recombinant porcine alpha interferon can reach 70%.

The solution that contains recombinant protein of above-mentioned acquisition is regulated pH value to 7.4 with NaOH, cross anion-exchange chromatography post (HiTrap Q FF, Amersham Biosciences), the speed of sample with 0.3ml/ minute is added in the chromatography column, carry out linear gradient elution with the PBS damping fluid of pH7.4 and the NaCl of 1M with 1ml/ minute speed, detect under the 280nm wavelength with Ultraviolet Detector, collect the target elution peak.Membrane filtration degerming with 0.22 μ m.Target protein to purifying carries out the SDS-PAGE electrophoresis detection, the result as shown in Figure 5, wherein, swimming lane M is the low molecular weight protein (LMWP) standard, swimming lane 1 is the protein sample of purifying.The result shows that a protein band appears in the protein sample of purifying near 23kD, conform to expected results.

The antiviral Determination of biological activity of embodiment 2, recombinant protein

Determination of biological activity suppresses method with reference to three appendix X of the Pharmacopoeia of the People's Republic of China in 2005 C cytoactive; with PK15 cell (ATCC Number:CCL-33)-vesicular stomatitis virus (Vesicularstomatitis virus; VSV) (ATCC Number:VR-1238AF) system; adopt CPE (cell cause a disease change effect) to suppress the inhibition micromethod for the basis, examining the inverse that product still can protect half cell (50%) to avoid the high dilution of virus attack with every milliliter of Interferon, rabbit is 1 interferon activity unit.

Concrete grammar is: get the PK15 cell in 96 orifice plates, be cultured to all adherent after, remove nutrient solution, be divided into two groups: one group with interferon alpha (available from the red Bioisystech Co., Ltd of Sichuan generation product, farming crude drug word (2003) 437730) as standard substance, by specification is diluted to 1000U/ml with interferon alpha, and every hole adds 0.1ml 4 times (4 ⁰, 4 ¹, 4 ², 4 ³, 4 ⁴4 ¹¹) doubling dilution liquid; Another is organized every hole and adds 0.1ml 4 times (4 ⁰, 4 ¹, 4 ², 4 ³, 4 ⁴4 ¹¹) embodiment 1 of doubling dilution obtains and recombinant protein (trial-product) solution of purifying.Use 100TCID ₅₀The vesicular stomatitis virus of dosage (VSV) is attacked poison, set up negative control (only to add standard substance or recombinant protein simultaneously through 4 times of doubling dilutions, do not add virus), positive control (do not add standard substance and recombinant protein, only add virus) and blank (do not add standard substance and recombinant protein, do not add virus), judged result in 50% pathology appears in the standard substance of observing 1U/ml under inverted microscope.Absorbancy is measured in the dyeing back at 570nm wavelength place, adopt four parametric regression computing methods to handle, and be calculated as follows interferon biological activity.3 repetitions are established in experiment.

Trial-product biologic activity=Pr * [(÷ of Ds * Es) (Dr * Er)]

Pr is interferon alpha standard substance biologic activity in the formula;

Ds is the pre-extension rate of trial-product;

Dr is the pre-extension rates of interferon alpha standard substance;

Es is the extension rate that trial-product is equivalent to interferon alpha standard substance median effective dose;

Er is that the interferon alpha standard substance are partly imitated extension rate.

The result shows that the recombinant protein activity that obtains is 1.6 * 10 ⁷U/mg ± 0.5 * 10 ⁷U/mg (mean+SD).

The Zadaxin Determination of biological activity of embodiment 3, recombinant protein

Adopt the Rose connection to measure the Zadaxin biologic activity, Zadaxin can activate takes off E acceptor thymocyte in the fresh pig thymus gland, makes it and sheep cell formation rosette.

1) gathers fresh pig thymus gland, preparation lymphocyte suspension;

2) 45 ℃ of temperature are bathed 1h (every jolting in 5 minutes once), and washed cell is also adjusted concentration to 3-5 * 10 ⁶Individual/ml, be positioned in the testing tube every pipe 0.2ml;

3) adding 0.1ml concentration in above-mentioned testing tube is 1 * 10 ^-3, 1 * 10 ^-4, 1 * 10 ^-5, 1 * 10 ^-6, 1 * 10 ^-7The recombinant protein of mg/ml, blank adds the sterilized water of 0.1ml, and 37 ℃ of temperature are bathed 1h;

4) cell concn is adjusted to 3-5 * 10 in sheep red blood cell (SRBC) washing back ⁷Individual/ml, each testing tube adds 0.2ml, shake up, and centrifugal 2 minutes of 600g, 4 ℃ of placements are spent the night;

5) next day abandoning supernatant, every pipe adds one of stationary liquid, shakes up gently, leaves standstill 10 minutes, adds 2 of staining fluids, shakes up, and leaves standstill counting after 15 minutes;

6) calculate in the field of microscope all lymphocytic numbers (being no less than 200) on 16 grids, statistics E rosette wherein forms number (in conjunction with the thymocyte of 3 above sheep red blood cell (SRBC)s), try to achieve the formation percentage, get its mean number, be sample and reference substance reading.Calculate the Zadaxin biologic activity by following formula.

Zadaxin biologic activity=testing sample E rosette percentage-blank E rosette percentage

Measurement result is as shown in table 2.

Table 2 recombinant protein Zadaxin biologic activity

Sample concentration (mg/ml)	Biological activity (%)
		Blank	0
1×10 -3	21
		1×10 -4	19
1×10 -5	15
		1×10 -6	7
1×10 -7	3

The safety experiment of embodiment 4, recombinant protein

(lot number is respectively 20060602 to get the 21 ages in days big York piglet of body condition health and the big York growing and fattening pigs of 5 monthly ages, 20060125, available from Shandong Province Academy of Agricultural Sciences breeding pig farm) each 20 (male and female half and half), be divided into 2 groups respectively at random, every group 10, totally 4 groups (piglet I group, piglet II group, growing and fattening pigs I group, growing and fattening pigs II group).The proof test of carry out as follows that single dose is once inoculated, single dose repeated inoculation and overdose being inoculated once.

Inoculation group of single dose: piglet I group and growing and fattening pigs I group are according to 1 dosage (10 of musculi colli injecting pathway inoculation ⁵The U/ head) embodiment 1 obtains the also recombinant protein of purifying, observes for 2 weeks continuously.

Single dose repeated inoculation group: will organize through piglet I group and the growing and fattening pigs I that single dose is once inoculated, after 2 weeks of inoculation, inoculate in the same way once, continue to observe for 2 weeks.

Inoculation group of overdose: piglet II group and growing and fattening pigs II group are according to 50 dosage (2 * 10 of musculi colli injecting pathway inoculation ⁷The U/ head) embodiment 1 obtains the also recombinant protein of purifying, observes for 2 weeks continuously.

Experimental session, viewing test animal clinical symptoms change comprised spirit, activity, breathes, searched for food, drinking-water, injection site has or not inflammatory reaction, drainage situation every day, every day take temperature, the reaction of record animal difference.If any death, dead pig is cutd open inspection, observe pathological change, analyze reason.

Through observing continuously in 2 weeks and 4 weeks, clinical symptom to pig before and after the injection recombinant protein compares, find that inoculation group of single dose, single dose repeated inoculation group and equal diet of inoculation group of overdose (50 dosage) are normal, the mental status does not have bad variation, breathe and drain no abnormality seen, the injection site does not have swelling and inflammation phenomenon, and experimental session does not find that animal has any untoward reaction.Whole experimental session, not seeing has pig death.The animal heat discovery is measured every day in the injection back, and most of pig inoculation back body temperature is normal, maintains the scope between 39.1-39.7 ℃, can the fervescence phenomenon occur second day of inoculation once in a while, but the time length is no more than 2 days.The result shows that the recombinant protein of embodiment 1 preparation is very high to the security of pig, with overdose (50 times of normal doses) injection, does not have remarkable toxic side effect, is a kind of safe novel antiviral preparation.

The clinic trial of embodiment 5, recombinant protein

1, the treatment of porcine reproductive and respiratory syndrome (blue otopathy)

The clinical identification of learning from else's experience is to suffer from the big York of the 21 ages in days piglet of porcine reproductive and respiratory syndrome, 70 age in days Du Luoke child care pigs, big York of 5 monthly ages to breed each 10 of pigs (lot number is respectively 20060302,20060120,20051001, available from Shandong Province Academy of Agricultural Sciences breeding pig farm), be divided into treatment group and control group, 5 of every group of every boars, every group totally 15.The treatment group presses 5 * 10 every day ⁴The recombinant protein that the dosage of U/ head injection embodiment 1 obtains, control group is only injected the physiological saline (placebo) of equal volume, injects continuously three days, observes every day and clinical symptom is marked, and concrete code of points is as shown in table 3, and the result is as shown in table 4.The result shows, there are notable difference in treatment group and the scoring of control group clinical symptom statistics, the recombinant protein of embodiment 1 preparation can be treated porcine reproductive and respiratory syndrome effectively, the morbidity swine disease feelings for the treatment of are clearly better, being embodied in the white corpuscle number rises to normal or near normal, heating, vomiting, symptom of diarrhea disappear; And the control group symptom of injection placebo does not have improvement.Mark in the table 4 is three days a mean value.

Table 3. clinical symptom code of points

Symptom	Mark
		Dead
	20

Anus temperature≤37 ℃ 37.1-39.4 ℃ 〉=39.5 ℃	1 2 3
		Mental status (consciously, great-hearted) tired out dispirited (consciously, but unvital) stupor (unconscious)	1 3 4
Dehydration 5% (slight---skin begins to occur fold) 8% (medium---there has been fold in skin) 12% (serious---shock)	1 3 4
		Ight soil proterties liquid state (diarrhoea) bloody stool	2 4
Stomachache is slight serious	1 2
		Vomiting	3

According to last table treatment group and group pig are given a mark, mark is high more, shows that body condition is poor more, and pig death is represented in full marks 20 timesharing.

The clinical symptom scoring statistics of table 4. treatment porcine reproductive and respiratory syndrome (blue otopathy)

Group	The treatment number of components		The control group mark
					The pig individuality	Before the treatment	After the treatment	Before the treatment	Behind the injection placebo
1	10	0	9	5
					2	17	20	18	20
3	13	5	13	20
					4	15	9	14	20
5	19	20	18	20
					6	18	20	19	20
7	12	6	11	8
					8	16	5	14	20
9	13	3	14	20

10	12	0	13	15
					11	9	0	8	13
12	11	2	10	15
					13	14	8	15	20
14	16	20	15	20
					15	12	3	12	20

2, the treatment of porcine epizootic diarrhea

The clinical identification of learning from else's experience is to suffer from the big York of the 21 ages in days piglet of porcine epizootic diarrhea, 70 age in days Du Luoke child care pigs, big York of 5 monthly ages to breed each 10 of pigs (lot number is respectively 20060302,20060120,20051001, available from Shandong Province Academy of Agricultural Sciences breeding pig farm), be divided into treatment group and control group, 5 of every group of every boars, every group totally 15.The treatment group presses 5 * 10 every day ⁴The recombinant protein that the dosage of U/ head injection embodiment 1 obtains, control group is only injected isopyknic physiological saline (placebo), injects continuously three days, observes every day and clinical symptom is marked, and concrete code of points is as shown in table 3, and the result is as shown in table 5.The result shows, there are notable difference in treatment group and the scoring of control group clinical symptom statistics, the recombinant protein of embodiment 1 preparation can be treated Porcine epidemic diarrhea virus effectively and be infected, the morbidity swine disease feelings for the treatment of are clearly better, being embodied in the white corpuscle number rises to normal or near normal, heating, vomiting, symptom of diarrhea disappear; And the control group symptom of injection placebo does not have improvement.

Mark in the table 5 is three days a mean value.

The clinical symptom scoring statistics of table 5. treatment porcine epizootic diarrhea

Group	The treatment number of components		The control group mark
					The pig individuality	Before the treatment	After the treatment	Before the treatment	Behind the injection placebo
1	15	18	16	20
					2	13	0	13	17
3	17	20	16	20
					4	14	2	12	19
5	13	0	13	17
					6	11	3	12	2
7	16	4	15	20
					8	15	2	16	18
9	10	0	11	17
					10	12	2	12	10

11	18	20	16	20
					12	10	0	13	16
13	11	2	10	3
					14	14	3	14	20
15	12	0	10	0

Sequence table

<160>4

<210>1

<211>204

<212>PRT

＜213〉artificial sequence

<400>1

Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu

1 5 10 15

Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly Gly Gly Gly

20 25 30

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

35 40 45

His Thr Arg Ala Leu Arg Leu Leu Ala Gln Met Arg Arg Ile Ser Pro

50 55 60

Phe Ser Cys Leu Asp His Arg Arg Asp Phe Gly Phe Pro Gln Glu Ala

65 70 75 80

Leu Gly Gly Asn Gln Val Gln Lys Ala Gln Ala Met Ala Leu Val His

85 90 95

Glu Met Leu Gln Gln Thr Phe Gln Leu Phe Ser Thr Glu Gly Ser Ala

100 105 110

Ala Ala Trp Asp Glu Ser Leu Leu His Gln Phe Cys Thr Gly Leu Asp

115 120 125

Gln Gln Leu Arg Asp Leu Glu Ala Cys Val Met Gln Glu Ala Gly Leu

130 135 140

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

145 150 155 160

Tyr Phe His Arg Leu Thr Leu Tyr Leu Gln Glu Lys Ser Tyr Ser Pro

165 170 175

Cys Ala Trp Glu Ile Val Arg Ala Glu Val Met Arg Ser Phe Ser Ser

180 185 190

Ser Arg Asn Leu Gln Asp Arg Leu Arg Lys Lys Glu

195 200

<210>2

<211>615

<212>DNA

＜213〉artificial sequence

<400>2

agtgacgcgg ctgtggatac tagctctgag attaccacca aagatctgaa agagaaaaaa 60

gaagttgtcg aagaagctga aaacggcggg ggtggatctg gtggcggggg aagctgtgac 120

ctgcctcaga cccacagcct ggctcacacc agggccctga ggctcctggc acaaatgagg 180

agaatctccc ccttctcctg cctggaccac agaagggact ttgggttccc ccaagaggcc 240

ttggggggca accaggtcca gaaggctcaa gccatggctc tggtgcatga gatgctccag 300

cagaccttcc agctcttcag cacagagggc tcggctgctg cctgggatga gagcctcctg 360

caccagttct gcactggact ggatcagcag ctcagggacc tggaagcctg tgtcatgcag 420

gaggcggggc tggaagggac ccccctgctg gaggaggact ccatcctggc tgtgaggaaa 480

tacttccaca gactcaccct ctatctgcaa gagaagagct acagcccctg tgcctgggag 540

atcgtcaggg cagaagtcat gagatccttc tcttcctcca gaaacctgca agacagactc 600

aggaagaagg agtaa 615

<210>3

<211>84

<212>DNA

＜213〉artificial sequence

<400>3

agtgacgcgg ctgtggatac tagctctgag attaccacca aagatctgaa agagaaaaaa 60

gaagttgtcg aagaagctga aaac 84

<210>4

<211>501

<212>DNA

＜213〉artificial sequence

<400>4

tgtgacctgc ctcagaccca cagcctggct cacaccaggg ccctgaggct cctggcacaa 60

atgaggagaa tctccccctt ctcctgcctg gaccacagaa gggactttgg gttcccccaa 120

gaggccttgg ggggcaacca ggtccagaag gctcaagcca tggctctggt gcatgagatg 180

ctccagcaga ccttccagct cttcagcaca gagggctcgg ctgctgcctg ggatgagagc 240

ctcctgcacc agttctgcac tggactggat cagcagctca gggacctgga agcctgtgtc 300

atgcaggagg cggggctgga agggaccccc ctgctggagg aggactccat cctggctgtg 360

aggaaatact tccacagact caccctctat ctgcaagaga agagctacag cccctgtgcc 420

tgggagatcg tcagggcaga agtcatgaga tccttctctt cctccagaaa cctgcaagac 480

agactcagga agaaggagta a 501

Claims (10)

1. albumen is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have Interferon, rabbit and Zadaxin active by (a) deutero-protein.

2. the described proteic encoding gene of claim 1.

3. according to the described proteic encoding gene of claim 2, it is characterized in that: described proteic encoding gene is following 1) or 2) gene:

1) its nucleotide sequence is a sequence 2 in the sequence table;

2) nucleotide sequence of the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 2.

4. the recombinant expression vector, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.

5. method of expressing the described recombinant protein of claim 1, be that claim 2 or 3 described recombinant protein genes are imported host cell by expression vector, screening obtains expressing the reconstitution cell of the described recombinant protein of claim 1, cultivates this reconstitution cell, expresses to obtain described recombinant protein.

6. method according to claim 5 is characterized in that: described expression vector is pBV220, pET carrier, pQE carrier or pPIC9K, and described host cell is bacillus coli DH 5 alpha, TB1 or BL21 (DE3).

7. method according to claim 6 is characterized in that: described expression vector is pBV220, and described host cell is a bacillus coli DH 5 alpha.

8. method according to claim 5 is characterized in that: the culture condition of described reorganization bacterium is 42 ℃ of abduction delivering 6-8 hours.

9. with the described recombinant protein of claim 1 medicine that prevents and/or treats swine disease poison infectious diseases of activeconstituents.

10. medicine according to claim 9 is characterized in that: described swine disease poison infectious diseases is porcine reproductive and respiratory syndrome, porcine epizootic diarrhea, transmissible gastroenteritis, rotavirus infection associated diseases, swine fever, PCV-II II type infection associated diseases, pseudoabies, porcine influenza, hydroa or parvovirus.

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