CN108794635B

CN108794635B - Bovine lactoferricin-human lysozyme fusion protein, gene and application thereof

Info

Publication number: CN108794635B
Application number: CN201810300197.9A
Authority: CN
Inventors: 孙杰; 汪钊; 魏春
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-04-04
Filing date: 2018-04-04
Publication date: 2021-03-19
Anticipated expiration: 2038-04-04
Also published as: CN108794635A

Abstract

The invention discloses a bovine lactoferrin peptide-human lysozyme fusion protein which is characterized in that the fusion protein is formed by sequentially connecting porcine myofibrillar protein antioxidant peptide, bovine lactoferrin peptide, chicken ovalbumin antioxidant peptide, porcine myotropomyosin antioxidant peptide, flexible connecting peptide and human lysozyme in series. Through fusion expression of the animal-derived anionic antioxidant peptide, the positive charge of the antibacterial peptide is counteracted, and the inhibition of the antibacterial peptide on host bacteria is reduced; the antioxidant peptide is beneficial to increasing the storage stability of the antibacterial peptide, and the antibacterial activity of the fusion protein to escherichia coli (ATCC 25922) is only reduced by 7.7% within 20 days after the fusion protein is placed at 4 ℃. Its viability to E.coli (ATCC 25922) was lost only 15.4% after 30 days.

Description

Bovine lactoferricin-human lysozyme fusion protein, gene and application thereof

(I) technical field

The invention relates to bovine lactoferricin-human lysozyme, a gene and application thereof.

(II) background of the invention

Abuse of antibiotics in the medical and animal farming fields causes an epidemic of drug-resistant bacteria, which poses a serious threat to human health. There is an urgent need to develop a novel antibacterial agent or feed additive to replace antibiotics. Antibacterial peptides and lysozyme are natural polypeptides and proteins with bactericidal activity, and are widely applied to clinical medicine and food industries as antibiotic substitutes.

The antibacterial mechanism of the antibacterial peptide is that the antibacterial peptide with positive charge is combined with the bacterial cell membrane with negative charge, and the bacterial death is caused by causing membrane perforation or inhibiting the physiological metabolic process of the bacteria. The N-terminal of bovine lactoferrin contains two antibacterial domains Lactoferricin (LFC) 17-30 and Lactoferricin (LFA) 265-284 generated by protease digestion. Lysozyme exerts its bactericidal effect by breaking down the beta- (1,4) glycosidic bond of peptidoglycan on cell walls. The lactoferrin antibacterial peptide and lysozyme have good thermal stability, particularly under the acidic condition, the antibacterial activity of the lactoferrin antibacterial peptide and lysozyme is not influenced by heating and even high-temperature and high-pressure treatment, and the lactoferrin antibacterial peptide and lysozyme are good substitutes for traditional antibiotics.

The Chinese invention patent CN101649311B discloses a preparation method of human lysozyme-antibacterial peptide fusion protein, which adopts a genetic engineering method to construct human lysozyme-antibacterial peptide recombinant plasmid, and then expresses the human lysozyme-antibacterial peptide fusion protein in eukaryotic cells. However, since lysozyme and antimicrobial peptides usually have positive cationic charges and may interact with DNA having anions, they have certain cytotoxicity to genetically engineered host bacteria, and are usually expressed in the form of fusion proteins to reduce the cytotoxicity. The Chinese invention patent CN105061603A discloses a preparation method of a hybrid protein of an antibacterial peptide thanatin protein, a 3GSA flexible peptide and a T4 lysozyme protein, which comprises the steps of carrying out fusion expression on the hybrid protein and a Sumo label in escherichia coli, then purifying the fusion protein, and cutting off the Sumo label to obtain the target antibacterial peptide-lysozyme fusion protein. The DNA sequence of fusion protein of polycation antibacterial peptide and polyanion peptide is artificially synthesized by Liqing and Zhouxiao macro (food science 2013) and is expressed in yeast. However, the fusion protein used in the two methods does not help the function of the final product, and the obtained product needs post-treatment and is too high in purification cost.

Disclosure of the invention

The invention aims to provide a lactoferrin peptide-human lysozyme hybrid protein and application thereof, and the product does not need to be cracked, and has stronger antibacterial activity and storage stability than lactoferrin peptide and human lysozyme.

The technical scheme adopted by the invention is as follows:

the invention provides a lactoferrin peptide-human lysozyme fusion protein which is formed by connecting pig myofibrillar protein antioxidant peptide (SEQ ID No.1), cow milk ferritin peptide (SEQ ID No.4), chicken ovalbumin antioxidant peptide (SEQ ID No.2), pig myotropomyosin antioxidant peptide (SEQ ID No.3), flexible connecting peptide (SEQ ID No.7) and human lysozyme (SEQ ID No.5) in series in sequence, wherein a DPconnecting sequence is connected between the pig myofibrillar protein antioxidant peptide (SEQ ID No.1) and the cow milk ferritin peptide (SEQ ID No.4), a NGDPE sequence is connected between the cow milk ferritin peptide (SEQ ID No.4) and the chicken ovalbumin antioxidant peptide (SEQ ID No.2), and an EDPNG sequence is connected between the flexible connecting peptide (SEQ ID No.7) and the human lysozyme (SEQ ID No.5) (a in figure 1). NGDPE and EDPNG between the above sequences are both amino acid sites susceptible to hydrolysis by acids, hydroxylamines and pepsin.

Further, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 6.

The invention also provides a bovine lactoferrin peptide-human lysozyme fusion protein coding gene, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 8.

In addition, the invention also relates to application of the bovine lactoferrin peptide-human lysozyme fusion protein in preparation of an antibacterial feed additive.

Further, the antibacterial feed additive is a livestock feed additive, and more preferably, the antibacterial feed additive is a piglet feed additive.

Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:

(1) through fusion expression of the animal-derived anionic antioxidant peptide, the positive charge of the antibacterial peptide is counteracted, and the inhibition of the antibacterial peptide on host bacteria is reduced; the antioxidant peptide is beneficial to increasing the storage stability of the antibacterial peptide, and the antibacterial activity of the fusion protein is only reduced by 7.7 percent within 20 days after the fusion protein is placed at 4 ℃. Its viability to E.coli (ATCC 25922) was lost only 15.4% after 30 days.

(2) The fusion protein of the bovine lactoferrin peptide and the human lysozyme is obtained by expressing the pichia pastoris, and the fusion protein has antibacterial activity without cracking, and has stronger antibacterial activity and wider antibacterial spectrum compared with the bovine lactoferrin peptide and the human lysozyme.

(3) The fusion protein sequence contains sites which are easily hydrolyzed by acid and pepsin, and after the pepsin is hydrolyzed in vitro, the bacteriostatic activity of the fusion protein to escherichia coli and staphylococcus aureus is respectively improved by 38.9 percent and 18.5 percent, so that the bacteriostatic activity of the fusion protein as a feed additive is enhanced after the fusion protein enters the digestive tract of animals and is cracked.

(4) The pichia pastoris product containing the fusion protein can improve the growth performance of piglets and reduce the diarrhea rate of the piglets.

(IV) description of the drawings

FIG. 1 shows the construction of the fusion protein sequence (a) and the plasmid (b). In a, seq.1 and 3 are pig myofibrillar protein antioxidant peptides, seq.4 is bovine lactoferrin peptide, seq.2 is chicken ovalbumin antioxidant peptide, seq.7 is flexible connecting peptide, seq.5 is human lysozyme sequence, and NGDPE and EDPNG between the sequences are amino acid sites which are easily hydrolyzed by acid, hydroxylamine and pepsin.

FIG. 2 shows the inhibition zone of the fermentation product of example 1. a is an escherichia coli (ATCC 25922) inhibition zone, and b is a staphylococcus aureus (ATCC 25923) inhibition zone. 1 recombinant bacteria fermentation liquor containing empty plasmid pGAPZ alpha; pastoris GS115 strain broth; 3 and 4, fermentation liquor of the recombinant bacteria;

FIG. 3 is a SDS-PAGE electrophoresis of samples at different fermentation time points.

FIG. 4 is a chromatogram of cycle preparation after lysis and dialysis, peak 1 being uncleaved LfcinB-hLY fusion protein, peak 2 being human lysozyme (hLY), and peak 3 being bovine lactoferrin peptide (LfcinB).

FIG. 5 shows Tricine-SDS-PAGE protein gels from different samples. M: marker; 1: electrophorogram of the sample before lysis, i.e. peak No.1 of fig. 3; 2: FIG. 3 is an electrophoretogram of peak 2 eluate; 3: FIG. 3 shows the electrophoresis of the peak 3 eluate.

FIG. 6 the zone of inhibition diameter of the LfcinB-hLY fusion protein against E.coli (ATCC 25922) after treatment at different temperatures and times.

FIG. 7 LfcinB-hLY fusion protein was shown to be bacteriostatic in E.coli (ATCC 25922) after treatment at different pH.

FIG. 8 shows the bacteriostatic activity of LfcinB-LhY fusion protein after treatment with different digestive enzymes.

FIG. 9 storage stability of LfcinB-hLY fusion protein.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1 fusion expression of bovine lactoferrin peptide-human lysozyme hybrid protein (LfcinB-hLY) in Pichia pastoris

Pig myofibrillar protein antioxidant peptide (SEQ ID NO.1), DPNG connecting sequence, cow milk ferritin peptide (SEQ ID NO.4), NGDPE connecting sequence, chicken ovalbumin antioxidant peptide (SEQ ID NO.2), pig myotropomyosin antioxidant peptide (SEQ ID NO.3), flexible connecting peptide (SEQ ID NO.7), EDPNG connecting sequence and human lysozyme (SEQ ID NO.5) are sequentially connected in series to form cow milk ferritin peptide-human lysozyme hybrid protein (SEQ ID NO.6) (a in figure 1). According to the LfcinB-hLY hybrid protein gene coding sequence SEQ ID NO.8 after pichia pastoris codon optimization, the gene is handed over to a gene synthesis company for whole gene synthesis.

Cloning the DNA sequence of SEQ ID NO.8 to a plasmid pGAPZ alpha (b in figure 1), carrying out plasmid linearization by using a restriction enzyme Bln I, and then electrically transforming a Pichia pastoris (P.pastoris) GS115 strain (Invitrogen company) to obtain a recombinant strain containing the LfcinB-hLY hybrid protein gene. 100 mu L of the bacterial liquid of the recombinant bacteria is uniformly coated on a YPDS solid plate containing 100 mu g/mL Zeocin, and the YPDS solid plate is cultured for 2 days at the constant temperature of 30 ℃. The positive transformant GS-LfcinB-hLY confirmed by PCR was cultured in YPD medium at 30 ℃ for 24 hours as a seed culture medium. Inoculating the seed solution into a fermentation medium at an inoculum size of 10% (v/v), and culturing at 200rpm and 30 ℃ for 72 h. The fermentation broth supernatant was collected, assayed for bacteriostatic activity, and the expression product was detected using 10% SDS-PAGE. Under the same conditions, the supernatant of plasmid pGAPZ alpha electrotransfer P.pastoris GS115 without LfcinB-hLY hybrid protein gene and the supernatant of P.pastoris GS115 fermentation without LfcinB-hLY hybrid protein gene and plasmid pGAPZ alpha are used as controls.

YPDS solid plate Mass end concentration composition: 1% yeast powder, 2% peptone, 2% glucose, 18.2% sorbitol, 2% agar powder, solvent deionized water, and natural pH value. When the Pichia host containing the target gene is cultured, Zeocin resistance with the final concentration of 100 mug/mL is added.

YPD medium composition at final concentration by mass: 1% yeast powder, 2% peptone, 2% glucose, deionized water as solvent, and natural pH value.

The final concentration of the fermentation medium is as follows: 5% glucose, 0.5% peptone, 1% NH₄AC, 2% yeast powder, 0.3% KH₂PO₄，0.03％MgSO₄，0.05％MnSO₄，0.05％CaCl₂The solvent is deionized water, and the pH value is natural.

And (3) detecting the bacteriostatic activity of the fusion protein: escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) are respectively selected to be singly colonized in LB liquid culture medium, and are subjected to shake culture at 37 ℃ and 200rpm for 12-16h, then OD600 is taken out and adjusted by sterile water to enable the OD600 of the bacterium liquid to be about 0.6. Adding the solid LB culture medium which is not solidified into a culture medium according to the bacterial liquid: LB medium ═ 1: adding E.coli bacteria liquid in a volume ratio of 1000, shaking gently, pouring into sterilized flat plate rapidly, pouring about 25mL, and standing for 20-30min to solidify. After coagulation, 4 holes were punched in the medium at equal distances along the mid-point of the medium radius using a sterilized 8mm diameter punch. 100uL of fermentation broth supernatant with protein concentration measured by a Coomassie brilliant blue method is added into holes of a culture medium, and then the culture medium is placed in a constant-temperature biochemical incubator at 37 ℃ for 8-12 h. Each sample was plated on 3 plates, and another plate was blanked with sterile water.

As shown in figure 2, neither the empty plasmid subjected to electrotransformation nor the supernatant of the P.pastoris GS115 strain fermentation broth showed antibacterial activity, the GS-LfcinB-hLY recombinant plasmid strain fermentation supernatant showed antibacterial activity against Escherichia coli and Staphylococcus aureus, and the diameters of the inhibition zones were 15mm and 25mm, respectively. And (3) concentrating fermentation samples at different time points (72h, 68h, 64h, 56h, 52h, 44h, 36h and 28h) by adopting a methanol precipitation method to obtain SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis images of the different sampling points, wherein suspected strips can be gradually seen after the fermentation is carried out for 64h, and are gradually deepened along with the increase of time, so that the activity of the detected antibacterial peptide is positively correlated, and the expression of the foreign protein is indicated.

The method for measuring the antibacterial potency of the fusion protein is dilution 2ⁿIf the time is no zone of inhibition, the potency AU: 2ⁿX 0.1 x 1/c, where n is the dilution of the minimum inhibitory concentration, 0.1 is the volume of fusion protein added, 0.1mL, and c is the concentration of fusion protein in mg/mL. The titer unit is AU/mg. Molar titers were defined as: AU/M. Where M is the relative molecular mass (kD) of the sample. Inoculating the recombinant strain seed liquid to a 5L fermentation tank containing 3L fermentation medium according to the inoculation amount with the volume concentration of 8%. The dissolved oxygen concentration was set at 20%, and 24 hours after inoculation, 10mL/h of the feed medium was added. And after 72h, ending fermentation until the bacterial mass is not increased any more. The final concentration of the feed medium is 5% glucose, 2% yeast powder and deionized water, and the pH value is natural. The expression amount of the exogenous protein is measured to be 35mg/L, the diameter of the supernatant inhibition zone of the fermentation liquor reaches 41mm, and the titer is 11.66 AU/mg.

EXAMPLE 2 isolation and purification of fusion protein LfcinB-hLY

(1) The fermentation broth obtained in example 1 was centrifuged and purified by means of an ion exchange resin SP Sepharose Fast Flow at a column volume of 20mL and UV detection wavelengths of 220nm and 280 nm. The column was equilibrated with 5mM acetate-sodium acetate buffer (pH 4.5) at a flow rate of 1.5 mL/min. 50mL of the fermentation broth supernatant was loaded. After loading was complete, the column was again equilibrated with the above buffer. After completion of the equilibration, elution was carried out using 50mM acetic acid-sodium acetate buffer (pH 4.5) containing 0.2mol/L NaCl as an eluent at a flow rate of 1.5 mL/min. After all elution peaks are collected, the antibacterial activity is measured, and a fusion protein LfcinB-hLY sample with the antibacterial activity is obtained. The elution peak with antibacterial activity obtained after the cation exchange chromatography is frozen, dried and concentrated to 1mL, namely the fusion protein LfcinB-hLY crude product with the titer of 17.62 AU/mg. The product was further purified on the one hand and cleaved on the other hand from the hydroxylamine hydrochloride of example 3.

(2) Further purifying by a circulating preparative chromatograph, wherein the filler is JAIGEL-GS 310; the sample injection amount is 10 mL; the flow rate is 5.0 mL/min; the UV detection wavelength was 280 nm. The mobile phase was 5mmol/L sodium acetate (pH 4.5). And (4) after collecting all elution peaks, measuring the antibacterial activity to obtain an elution peak with the antibacterial activity. The antibacterial potency of the elution peak is 39.68AU/mg, namely the fusion protein LfcinB-hLY pure product.

EXAMPLE 3 cleavage of the fusion protein LfcinB-hLY with hydroxylamine hydrochloride

Hydroxylamine lysis buffer 25.80g hydroxylamine hydrochloride and 4.8g Tris dissolved in 100mL double distilled water, using 4M NaOH to adjust the pH value to 9.0 and fixed to 200 mL.

The fusion protein sequence is designed with Nordrin hydroxylamine cleavage site (Asn-Gly). To the fusion protein LfcinB-hLY sample obtained by the cation exchange chromatography in step (1) of example 2, a hydroxylamine lysis buffer was added in an amount twice the volume thereof, and after being subjected to a water bath at 45 ℃ for 4 hours, the reaction solution was adjusted to pH 7 with hydrochloric acid and the reaction temperature was lowered to room temperature, thereby terminating the lysis reaction. Hydroxylamine and small peptides were then removed using a 1KD dialysis bag. The dialyzed permeate was purified by preparative chromatograph, and the operation was the same as in example 2. As shown in FIG. 4, the absorption peak is No.1 at around 21min, No.2 at around 24min, and No.3 at around 28 min. According to the principle of separating polypeptide by a circulating preparation chromatograph, three absorption peaks respectively correspond to uncleaved LfcinB-hLY fusion protein, human lysozyme (hLY) and bovine lactoferrin peptide (LfcinB).

The eluates with three absorption peaks were concentrated and then assayed for antibacterial activity. The eluent of the absorption peak No.1 is uncleaved LfcinB-hLY fusion protein, the absorption peak No.2 is hLY, and the absorption peak No.3 is LfcinB. The potency of the fusion protein LfcinB-hLY to inhibit e.coli K88 before and after lysis was determined as in example 1. The results of the assay showed that the antibacterial potency of LfcinB-hLY (39.68AU/mg), hLY (17.3AU/mg), LfcinB (6.6AU/mg), and fusion protein LfcinB-hLY was higher than that of hLY and LfcinB alone, respectively.

Collecting eluate of the circulating preparative chromatography, and performing Tricine-SDS-PAGE protein electrophoresis on a protein sample after methanol precipitation and concentration. The methanol precipitation method of the protein comprises the steps of adding 4 times of precooled pure methanol, 2 times of chloroform and 3 times of precooled distilled water into the protein to be precipitated, violently mixing the mixture evenly, standing the mixture for 30min at 4 ℃, and centrifuging the mixture for 1min at 8600 g. The upper aqueous phase was removed. The lower layer and the intermediate protein precipitate were mixed well with 1/2 volumes of pre-cooled methanol and centrifuged at 9000g for 5 min. The supernatant was discarded, dried, and the protein precipitate was dissolved using loading buffer. As shown in FIG. 5, the band No.1 is a sample of the polypeptide with the absorption peak No.1 in FIG. 4, i.e., a sample of the uncleaved fusion protein LfcinB-hLY, and has a size of about 24 kD. The band No.2 is a polypeptide sample of the absorption peak No.2 in FIG. 4, and the size is about 14kD, which is equivalent to the molecular weight of human lysozyme. The band 3 is a polypeptide sample of the absorption peak No.3 in FIG. 4, and has a size of about 4kD, which is equivalent to the size of bovine lactoferrin peptide.

EXAMPLE 4 Studies of the Properties of the fusion protein LfcinB-hLY

1. Study on bacterial inhibition spectrum of LfcinB-hLY fusion protein

The bacteriostatic activity of the LfcinB-hLY fusion protein on the strains listed in Table 1 was tested. Each of the bacteria was cultured in 5mL of liquid medium by shaking culture under the conditions shown in Table 1. When the bacteria grow to the logarithmic growth phase, the inhibition zone is determined according to the method of example 1. The measurements were averaged 3 times.

The LB culture medium comprises tryptone 1%, yeast extract 0.5%, NaCl 1%, and deionized water as solvent, and has natural pH.

The amount of the Chao's culture medium is 3 percent of sucrose, 0.3 percent of sodium nitrate, 0.1 percent of dipotassium phosphate, 0.05 percent of magnesium sulfate, 0.05 percent of potassium chloride and 0.001 percent of ferrous sulfate, the solvent is deionized water, and the pH value is natural.

MRS medium was purchased from OXOID.

The LfcinB-hLY fusion protein has bacteriostatic effects on gram-positive bacteria represented by staphylococcus aureus, bacillus subtilis and bacillus licheniformis and gram-negative bacteria represented by escherichia coli, wherein the effect on the staphylococcus aureus is most obvious. Has no inhibiting effect on mould represented by aspergillus oryzae, no inhibiting effect on saccharomyces cerevisiae, and strong inhibiting effect on candida of the same eukaryote. Therefore, unlike lysozyme which has strong bacteriostatic activity only on gram-positive bacteria, the LfcinB-hLY fusion protein has good bacteriostatic effect on gram-positive bacteria and gram-negative bacteria.

TABLE 1 culture conditions of different indicator bacteria and bacteriostatic effect of LfcinB-hLY fusion protein

Note: the diameter of the inhibition zone is larger than 20 mm; + shows that the bacteriostasis zone effect is more than 13 mm; and/means no bacteriostatic effect.

2. Stability of LfcinB-hLY fusion protein

2.1 thermal stabilization

The fusion protein LfcinB-hLY prepared in step (1) of example 2 was treated at 60 deg.C, 80 deg.C, 100 deg.C, 120 deg.C for 10min, 30min, 60min, respectively, and E.coli was used as indicator bacteria to test bacteriostatic activity, and untreated samples were used as control, and the results are shown in FIG. 6.

As can be seen from FIG. 6, the activity of the LfcinB-hLY fusion protein was not significantly reduced after 60min and 80 min treatment. The activity of the water bath heating at 100 ℃ for 10min is reduced by 2.8 percent, and the activity is reduced by 8.6 percent obviously when the water bath heating is carried out for 60min along with the increase of the heating time. The activity of the fusion protein is reduced by 8.6 percent when the fusion protein is heated in a sterilization pot for 10min at 120 ℃, the activity loss is the same as that of the fusion protein heated in boiling water for 60min, and the activity loss reaches 22.8 percent when the fusion protein is heated for 60min, so that the fusion protein has better thermal stability.

2.2 acid-base stability

The fusion protein LfcinB-hLY prepared in step (2) of example 2 was treated at pH3, 4, 5, 6, 7, 8, and 9 for 1h, E.coli was used as indicator bacteria, the zone diameter was measured, and untreated samples were used as control, and the results are shown in FIG. 7.

As shown in FIG. 7, the antibacterial activity was stronger at pH 4, and decreased in comparison with the control group under the acidic condition, and the antibacterial effect on Escherichia coli (ATCC 25922) showed a decrease with the increase of pH, and was zero when the pH was increased to 7. Therefore, the stability of the fermentation liquor is better when the natural pH value of the fermentation liquor is about 4.5.

2.3 stability of enzymatic hydrolysis

The solution of the fusion protein LfcinB-hLY prepared in step (2) of example 2 was added to the digestive enzyme at a final concentration of 1mg/mL, and adjusted with 0.1M HCl and 0.2M NaOH to give the pH values of Table 2. Treating for 1h under the conditions shown in Table 2, inactivating in boiling water bath for 5min, adjusting pH to about 5 with corresponding acid and base, determining inhibition zone with untreated fusion protein as control and Escherichia coli and Staphylococcus aureus as indicator bacteria, and finding the result shown in FIG. 8.

TABLE 2 reaction conditions for different enzymes

As can be seen from FIG. 8, pepsin has a promoting effect on the bacteriostatic activity of the LfcinB-hLY fusion protein on Staphylococcus aureus and Escherichia coli, and it is possible that the bacteriostatic activity of LfcinB and hLY released by pepsin hydrolysis has a synergistic effect. Trypsin and chymotrypsin had less effect on the antibacterial activity than the control group. The LfcinB-hLY fusion protein has strong tolerance to various proteases in animal gastrointestinal tracts.

2.4 storage stability of LfcinB-hLY-containing fusion proteins

The fusion protein LfcinB-hLY prepared in step (2) of example 2 and the mixture of peak 2 and peak 3 obtained in example 3 were tested for antibacterial activity at 4 ℃ for different periods of time using E.coli (ATCC 25922) as an indicator, and the protein concentrations in both solutions were equal and compared on day one. As seen in FIG. 9, the bacteriostatic activity of LfcinB-hLY fusion protein decreased by only 7.7% within 20 days of storage. After 30 days, the inhibitory activity of the strain on Escherichia coli (ATCC 25922) is only lost by 15.4%. The drop of the antibacterial activity of the hLY and LfcinB mixed solution obtained after hydroxylamine cracking and dialysis to remove the anti-small-molecule oxidized peptide is obviously faster than that of the LfcinB-hLY fusion protein, and the activity of the LfcinB mixed solution to escherichia coli is only lost by 38.8% after 30 days.

Example 5 high Density fermentation of Pichia pastoris for expression of LfcinB-hLY fusion protein

(1) Picking a single colony of the recombinant bacterium containing the LfcinB-hLY hybrid protein gene constructed in example 1 from a YPD plate, inoculating the single colony to 5mL of YPD medium, and carrying out shaking culture at 30 ℃ for overnight; mixing the raw materials in a ratio of 1: inoculating the bacterial liquid into 300mL YPD culture medium in a volume ratio of 100, and performing shaking culture at 30 ℃ overnight until OD600 reaches 2-6 to serve as seed liquid for inoculating a fermentation tank.

(2) A30L fermenter made in China was charged with 10L of the fermentation medium (composition as in example 1), sterilized at 121 ℃ for 20 minutes, adjusted to 30 ℃ and adjusted to pH 4.5 with ammonia water. Inoculating the seed liquid in the step (1) according to the inoculation amount with the volume concentration of 8%, controlling the temperature below 30 ℃ and the rotating speed between 300 and 800rpm in the fermentation process, and adjusting the pH value through a phosphoric acid solution and ammonia water while ensuring that the dissolved oxygen is not lower than 20%. 24 hours after inoculation, 10mL/h of feed medium (same composition as in example 1) was added. And after 72h, ending fermentation until the bacterial mass is not increased any more. Discharging materials after fermentation is finished, and spray drying the fermentation liquor to obtain the fusion protein LfcinB-hLY pichia pastoris product. In addition, the same fermentation was carried out on Pichia pastoris GS115 host cells which do not contain the LfcinB-hLY gene.

EXAMPLE 6 piglet feeding experiment with Pichia pastoris preparation containing fusion protein LfcinB-hLY

In order to verify the influence of the recombinant protein Lfcin B-hLY on the growth performance and immunity of piglets, a piglet feeding experiment of the recombinant protein Lfcin B-hLY is carried out. The experiment was divided into experiment group I, experiment group II and control group, each group having 12 heads. The experimental group and the control group select weaned piglets with similar age in days and weight. Feeding basal diet for the control group; experiment group I, the pichia pastoris product LfcinB-hLY is fed with the fusion protein with the mass concentration of 0.3 percent and the basic ration. Experiment group II, the basal diet + Pichia pastoris product (i.e., Pichia pastoris GS115 host strain fermentation product prepared in example 5) with a mass concentration of 0.3% and without fusion protein LfcinB-hLY was fed. The test results are as follows:

	experiment ofGroup I	Experimental group II	Control group
				Initial average weight/Kg	7.6	7.5	7.4
End average weight/Kg	24.6	22.9	21.8
				Days of experiment	45	45	45
Daily gain	377.8	342.2	320
				Meat ratio of materials	1.84	1.89	2.18
Survival rate	100％	100％	100％
				Rate of diarrhea	3.6％	5.6％	7.0％

The experimental results are as follows:

1. the feed is added with a pichia pastoris product, the daily gain of piglets is increased by 22.2 g compared with that of a control group, and when the pichia pastoris product containing the recombinant protein LFB-hLY is added, the daily gain of the piglets is further increased by 57.8 g compared with that of the control group, which indicates that the growth performance of the piglets is improved by the recombinant protein LFB-hLY;

2. the feed conversion ratio of the experimental group I is lower than that of the control group by 0.34, and the feed conversion ratio of the experimental group II is lower than that of the control group by 0.29, which shows that the feed utilization rate is improved by adding the pichia pastoris, and the feed utilization rate is slightly improved by adding the recombinant protein LFB-hLY;

3. the diarrhea conditions of the experimental group I and the experimental group II are both less than those of the control group, and the effect of the pichia pastoris product containing the recombinant protein LFB-hLY on preventing the diarrhea of the piglets is better than that of the pichia pastoris product without the recombinant protein LFB-hLY.

Sequence listing

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Ile Ser Leu Ala Asn Trp Met Cys Leu Ala Lys Trp Glu Ser Gly Tyr

115 120 125

Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly Asp Arg Ser Thr Asp Tyr

130 135 140

Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp Cys Asn Asp Gly Lys Thr

145 150 155 160

Pro Gly Ala Val Asn Ala Cys His Leu Ser Cys Ser Ala Leu Leu Gln

165 170 175

Asp Asn Ile Ala Asp Ala Val Ala Cys Ala Lys Arg Val Val Arg Asp

180 185 190

Pro Gln Gly Ile Arg Ala Trp Val Ala Trp Arg Asn Arg Cys Gln Asn

195 200 205

Arg Asp Val Arg Gln Tyr Val Gln Gly Cys Gly Val

210 215 220

<210> 7

<211> 15

<212> PRT

<213> Unknown (Unknown)

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 8

<211> 663

<212> DNA

<213> Unknown (Unknown)

<400> 8

gacgcccaag agaaactgga gatagaagca gaaggagaag accctaatgg ctggaaactt 60

ctttccaaag ctcaggaaaa attcggcaag aacaagagta gatttaagtg ctggagatgg 120

cagtggcgtt ggaaaaagtt aggtgcaaac ggtgatcctg aagatgaaga cactcaagct 180

atgccagaag aacttgacaa tgcacttaac ggtggtggtg gttcaggcgg cggaggatcc 240

ggcggcggcg gaagtgagga ccctaacggc aaagtcttcg agaggtgcga gcttgccagg 300

acgcttaaac gtttgggtat ggacggatac cgtggtatat cacttgcaaa ctggatgtgc 360

ttagcaaaat gggagagtgg atacaacact agagctacga attacaacgc tggtgacagg 420

tcaactgatt atggtatttt ccaaatcaat tccagatact ggtgtaatga cggcaaaacc 480

cctggtgccg tcaacgcctg tcatttaagt tgctctgcat tactacaaga caacatagcc 540

gatgctgtag cttgcgcaaa gagagttgtg agggatcctc aaggtatcag ggcatgggtg 600

gcttggagga acagatgtca aaatagggat gttcgtcaat atgttcaagg ttgtggagtc 660

tga 663

Claims

1. A bovine lactoferricin-human lysozyme fusion protein is characterized in that the amino acid sequence of the fusion protein is shown in SEQ ID NO. 6.

2. The bovine lactoferrin peptide-human lysozyme fusion protein coding gene of claim 1, wherein the nucleotide sequence of the coding gene is represented by SEQ ID No. 8.

3. Use of the bovine lactoferrin peptide-human lysozyme fusion protein of claim 1 in the preparation of an antibacterial feed additive, wherein the antibacterial species is bacteria or candida.

4. The use according to claim 3, wherein the antibacterial feed additive is a livestock feed additive.

5. The use according to claim 4, wherein the antibacterial feed additive is a piglet feed additive.