CN104418946A - Mytilin as well as preparation method and application thereof in preparation of medicines and feed additives - Google Patents

Abstract

The invention discloses a 'mytilin as well as a preparation method and an application thereof in preparation of medicines and feed additives', relating to bio-pharmaceutical technologies. The mytilin has an amino acid sequence shown by Seq ID No. 1. Verified by cell tests, the mytilin has a significant inhibition effect on hepatitis B virus, and verified by animal tests, the mytilin also has an effect of comprehensively improving the health conditions of animals. The invention also provides a scheme for preparing a polypeptide by using a gene engineering method, and by adopting the scheme, mytilin products with the level of more than gram grades can be prepared. Because the mytilin disclosed by the invention has the effects in the aspects of resisting virus, comprehensively improving the health conditions of the animals, and the like, the mytilin can be taken as a main component for preparing treatment medicines and health products which can be used for both human and animals, and can be applied to the treatment medicines, the health products and the feed additives.

Description

Training and peptide and preparing the application in medicine and fodder additives, preparation method

First part: technical field

The present invention relates to field of biological pharmacy, particularly relate to a kind of training and peptide and preparing the application in medicine, healthy products and fodder additives, preparation method.

Second section: background technology

Alexin is that the one produced through induction in organism has bioactive micromolecule polypeptide, natural immunity material, and molecular weight, about 3000 ~ 7000, is made up of 20 ~ 60 amino-acid residues, multiple disulfide linkage.This kind of active polypeptide majority has the features such as strong basicity, thermostability and broad-spectrum antimicrobial.Natural alexin is distributed in from plant, lower animal to the nearly all biological group such as Mammals, is the important component part of biological autoprotcrtive system.

Most alexin can effectively kill gram-negative bacteria and gram-positive bacteria.Concentration is that namely the alexin of 10 ~ 100mg/L has lethal effect to various bacteria in vitro, and the concentration of alexin in neutrophil leucocyte is g/L level, considerably beyond above-mentioned numerical value, this shows that alexin may have stronger fungicidal activity in vivo, and research at present finds that the kill capability of alexin to gram-positive bacteria is obviously better than gram-negative bacteria.In vitro, HBD-2 is 0.46nmol/ml to colibacillary medium lethal dose (LD50), and minimal inhibitory concentration (MIC) is 15 μ g/ml, and is 62 μ g/ml to the MIC of Pseudomonas aeruginosa, streptococcus aureus.Experiment in vitro proves that most of alexinic MIC scope is 0.5-10 μm of ol/L.

Alexin can also kill some tunicle viruses, as HIV, simplexvirus, bubble type Stomatovirus, but to invalid without capsid virus.θ-alexin also has anti-virus and toxinicide effect.Alexin is mainly through being combined with virus capsid protein thus causing viral loss of biological activity, and this special role mechanism also makes microorganism not easily produce resistivity to it.Alexin directly can suppress virus, the suppression degree of virus is depended on to the tightness degree of alexin concentration and intramolecular disulfide bond, its antiviral efficacy is equally by the impact of the factors such as time, pH value, ionic strength and temperature, under neutrality and conditions of low ionic strength, alexin has stronger antiviral activity.

Alexin not only directly can resist pathogenic micro-organism, but also has immunoregulation effect.The effect that alexin is transmitted by cell signal, strengthens non-specific immunity cell, especially the activity of scavenger cell and chemotaxis.Alexin can also promote chemotactic and the propagation of body T cell, enhancing body immunne response ability, regulates specific immunity, strengthens living organism Initiative Defense function.Alexin can start acquired immune system, and by innate immunity and the organic connection of acquired immunity as cell surface receptors such as a kind of effector molecule activating macrophage, DC, tracheal epithelial cells.Now prove that some α-alexins, beta-alexin have chemotactic activity to T cell, monocyte and immature DC, can induced monocyte and epithelial cell generation cytokine.The neutrophil leucocyte alexin of people, mouse, pig, rabbit can be induced mast cell degranulation and discharge histamine.Beta-alexin also by combining with human chemokine receptor 6 (CCR6), thus attracts jejune dendritic cell (DC) and memory T cell (Tm) to inflammation part, activating cells immunity and humoral immunization.In addition, alexin directly can also promote supplementing and gathering of infection site neutrophil leucocyte.

The use havoc of the Antibiotic Additive microecological balance of animal intestinal, drug residue also have impact on quality and the human health of livestock product, and derive from shellfish, mammiferous alexin relative molecular mass is less, thermostability and water-soluble all better, can absorb in enteron aisle.Because alexin belongs to polypeptide moiety, be easily easily degraded by proteases in vivo as amino acid, general noresidue in vivo after feed intake.Utilize gene engineering method production environment-friendly type alexin fodder additives or added by daily ration, all can effectively improve immunity, prevent disease and growth promoting effects, as reduced stillborn foetus, mummy and deformity; Improve birth weight and piglet birth uniformity coefficient; Raising weaned piglet is heavy, number of weaned, reduces nursery-age pig sickness rate; Reduce milking sow sickness rate, reduce mother, piglet is various stress; Improve sow subhealth state and breeding difficulty, extend sow working life; Improve child care pig surviving rate, reduce sickness rate; Improve child care daily gain in pigs, reduce feedstuff-meat ratio; Improve immunosuppression, improve immunne response ability, antibody horizontal is more neat.

Be derived from the alexin Mytilin B aminoacid sequence of Mediterranean Sea mussel as shown in Seq ID No.3, it is made up of 34 amino-acid residues, 4 disulfide linkage, composite screw and beta structures.Mytilin B alexin has broad spectrum of activity, can suppress Gram-positive and negative bacterium, has antiviral activity simultaneously.Mytilin B alexin utilize N hold with positive charge and the intensive negative electrostatic charge of the cell membrane phospholipid bilayer of cell of prokaryotic cell prokaryocyte attracting, the insertion hydrophobic region that C end is flexible, under amphipathic alpha-helix effect, prokaryotic cell prokaryocyte film is formed the ionic channel of 4nm, the formation of ionic channel changes the osmotic pressure inside and outside cytolemma, in cytolemma, material exosmoses in a large number, causes bacterial cell death, plays germicidal action.The alexinic antiviral-mechanism of Mytilin B is the capsid specific binding with virus, thus suppresses copying of virus.

French scientist has done a large amount of work in the alexin Mytilin research of mussel, natural mussel defence have Mytilin A (Seq ID No.8), Mytilin B (Seq ID No.3), Mytilin C (Seq ID No.9), Mytilin D (Seq ID No.10) and Mytilin G (Seq ID No.11) etc.Because alexin Mytilin molecule is little, there is certain difficulty in separating-purifying, and natural resource are limited, is difficult to industrialization; Because alexinic disulfide linkage and polypeptide space structure determine its basic characteristic, chemosynthesis can not obtain the product with excellent activity, and research shows in many ways, one of biologically active prod percentage only having natural product of chemosynthesis or lower; Only have genetically engineered approach could obtain the alexin with excellent activity, because high density Mytilin alexin polypeptide can kill most host cell, simultaneously in secreting, expressing process, alexin easily degrade by the enzyme system of Host Strains, thus produce internecine situation and cannot really obtain enough expression products.So, also there is no the engineering bacterium expression industrialization production technology of Mytilin alexin polypeptide at present, do not see and accurately express and obtain this enough polypeptide with the report be for experiment, not seen in its application report in treating hepatitis B, immune drug, fodder additives etc. yet.

Part III: summary of the invention

The present invention, according to the blank in above-mentioned field and demand, provides one be derived from the alexinic Altered peptide of Mediterranean Sea mussel (training and peptide) and preparing the application in medicine, healthy products and fodder additives, preparation method

Training and peptide is characterized in that having the aminoacid sequence shown in Seq ID No.1.

To encode the gene of above-mentioned training and peptide.

The nucleotide sequence of described gene is as shown in Seq ID No.2.

Expression vector containing said gene.

The skeleton carrier of described expression vector is pKLAC1.

The preparation method of above-mentioned training and peptide, is characterized in that above-mentioned expression vector to be transferred in yeast cell, fermentation culture.

Described yeast cell is K.lactis YCT799.

Above-mentioned training and peptide are preparing the application in medicine, healthy products and fodder additives, preparation method.

Described medicine and product are oral preparations.

The present invention is according to the alexin Mytilin B being derived from Mediterranean Sea mussel, homology degree 97%, for improving its manufacturing, carries out allosteric to its aminoacid sequence, increase G is held at its N, obtain the polypeptide as shown in Seq ID No.1, called after training and peptide, be made up of 35 amino acid, molecular weight is 4038.8Da, iso-electric point 9.58, hydrophobic ratio 40%, net charge+10.By allosteric, the polypeptide that secreting, expressing is gone out becomes stable, can not be degraded rapidly, thus achieve industrialized manufacture in fermentation, purifying and preservation process by proteolytic enzyme.By allosteric, add this polypeptide transformation period in animal body, more effective action effect can be obtained.The present invention, by aminoacid sequence allosteric, produceability and stability changes Mytilin B; Test proves, the training that the present invention adopts gene engineering method to prepare and the productive rate of peptide were more than 0.2% (referring to the ratio of expression product compared with the substratum quality of giving money as a gift that purifying obtains).Same aminoacid sequence allosteric, namely increase G is held at N, to obtain good manufacturing, also may be used for Mytilin A, the allosteric of Mytilin C, Mytilin D and Mytilin G and preparation, the aminoacid sequence after allosteric is respectively Seq ID No.4, Seq ID No.5, Seq ID No.6 and Seq ID No.7.These sequences can be used for the preparation of medicine, healthy products and fodder additives.

The aminoacid sequence of the present invention according to above-mentioned Seq ID No.1, devise the gene order of this aminoacid sequence of coding, utilize the degenerate of codon, have adjusted nucleotide sequence to make it meet yeast expression system to obtain the nucleotide sequence shown in SeqID No.2 to the Preference of codon, be conducive to stability and high efficiency and express alexin.

Present invention also offers the expression vector containing said gene, the skeleton carrier adopted is that the structure of pKLAC1 or pKLAC2, pKLAC2 is shown in Figure of description 1, and by pKLAC1 or pKLAC2, recombinant protein wherein can at cell inner expression, also can secreting, expressing.Be conducive to the alexin obtaining high yield and purity.

Present invention also offers training and the preparation method of peptide, the present invention is transferred in yeast expression system by building the expression vector obtained, and makes it express training and peptide, by follow-up separation and purification drying step obtain train and peptide sterling.The competent yeast cells K.lactis YCT799 that the present invention preferably adopts, non-nutritive defect body, does not have genetic marker yet, is applicable to the conversion of the expression vector based on linearizing pKLAC or pKLMF.Use this expression system, can guarantee that training and the peptide of secreting, expressing can not be degraded and possess activity further, this expression system also industrialization can express Mytilin A (Seq ID No.8), Mytilin B (Seq ID No.3), Mytilin C (Seq ID No.9), Mytilin D (Seq ID No.10) and Mytilin G (Seq IDNo.11), makes it can be used for medicine manufacture, healthy products and fodder additives.

Training provided by the invention and peptide as the main component of medicine, healthcare product and fodder additives, can be prepared into medicine and the product of various formulation.As hepatitis B medicine, antiviral, immunostimulant product, sleep improvement product, fodder additives, functional food, veterinary drug, growth stimulant etc.Drug testing in vitro shows, training and peptide have obvious restraining effect to hepatitis B virus activity, see embodiment 2.Animal experiment shows, it improves the effect of animal health status in addition comprehensively, sees embodiment 3.The present invention is preferentially prepared into oral preparations.

Part IV: accompanying drawing explanation

Fig. 1. skeleton carrier pKLAC1 structure iron

New England Biolabs, Inc provides

Fig. 2. the plasmid sequencer address of training prepared by the present invention and peptide

Enzyme is cut the clone of the correct recombinant plasmid pMD19-T of qualification, clone is entirely true in sequencing result display.

Part V: embodiment

The preparation of embodiment 1. training and peptide

Step 1. foreign DNA synthesizes

Utilize DNA chemosynthesis instrument to prepare foreign gene, obtain the exogenous dna fragment of coding training and peptide, as shown in Sequence IDNo.2.

Checking: cut by enzyme and identify that correct clone delivers to promise match genome research center, Beijing and carries out sequencing, sequencer address is shown in (Fig. 2).

The structure of step 2. expression vector

The pKLAC1 (being purchased from New England Biolabs, Inc) that expression vector provides for K.lactis protein expression test kit on pKLAC1 (9097bp), Lac4 promotor (P _lAC4-PBI) 5 ' and 3 ' to hold by the replication origin of the gene of encoding beta-lactamase (ApR) and pMB1 institute every Ji.K.lactis α-binding factor secretion homing sequence (α-MF), multiple clone site (MCS) and Lac4 transcription terminator (TT) is positioned at 3 ' P _lAC4-PBIdownstream; The P of yeast _aDH2the expression of regulation and control acetamidase marker gene (amdS).

Cut carrier pKLAC1 with Sac II or Bst XI enzyme and make its linearizing, exogenous dna fragment Sequence ID No.2 is inserted into the Lac4 promotor site of yeast K.lactis genome self, obtain recombinant vectors.In this recombinant vectors, goal gene is cloned into K.lactis α binding factor (the α Mating Factor on pKLAC1, α-MF) downstream, see Fig. 1, in the nucleus of yeast K.lactis, the DNA of coding for alpha-MF structural domain and foreign recombinant proteins and Yeast genome are integrated, and the α-MF of coding is sheared to make target protein matter secreting, expressing the most at last on golgi body.

P _lAC4-PBIpromoter regulation is expressed: once fused protein Ji begins to express, the signal peptide be positioned on α-MF will instruct fused protein to be transported on endoplasmic reticulum (ER) film, is excised by signal peptide at this signal peptidase (SP).Fused protein is transported to golgi body by secretory vesicle (annular), and α-MF structural domain excises at this by Kex proteolytic enzyme, the target protein matter that release is ripe.Subsequently, target protein matter is transported to plasma membrane (PM) by secretory vesicle, is secreted into born of the same parents from plasma membrane.

Step 2. transforms

Use K.lactis YCT799 (being purchased from New England Biolabs, Inc) competent cell.

Step 1 is built the recombinant vectors obtained and is placed in competent cell solution, make recombinant plasmid be adsorbed in the surface of competent cell, ice bath for some time, cytolemma is in contraction schedule.Then rapid competent cell is placed in 42 DEG C, makes cell membrane channel in thermal environment open, recombinant plasmid by the outer high concentration region of film to film internal diffusion, after 45 second time, put back in ice bath by competent cell again, membrane channel is closed, and recombinant plasmid proceeds to yeast.

Yeast transformant ethanamide screens, and is applied on the basic carbon source of yeast (YCB) substratum containing 5mM ethanamide by cell transformation mixture (containing being transformed by pKLAC1 in a large number or unconverted cell).YCB substratum contains all nutritive substances needed for K.lactis Growth of Cells and carbon source, but lacks nitrogenous source.Only have when ethanamide is degraded to ammonia by acetamidase (the amdS genetic expression by pKLAC1), could be utilized as nitrogenous source.Therefore, the cell of conversion is only had just can to grow up to bacterium colony.

Checking:

Picking colony, with shake-flask culture, substratum is the YPGal substratum (1% yeast extract, 2% peptone, 2% semi-lactosi) containing 38mM ammonium sulfate, 28 DEG C ~ 30 DEG C, incubation time 70 hours.Centrifugal 3000rpm, gets supernatant liquor, agarose gel electrophoresis analysis.

The cultivation of step 3. transformant

Containing YPGal substratum (2% yeast extract of 38mM ammonium sulfate, 4% peptone, 6% semi-lactosi), standard fermentor deep ventilation is cultivated, and 120 DEG C of wet-hot steam sterilizings 30 minutes, are cooled to 28 DEG C, inoculation, 28 DEG C ~ 30 DEG C, in fermenting process, stream mends semi-lactosi 6% again, incubation time 70 hours.

The separation and purification of step 4 training and peptide

Fermented liquid adopts ceramic membrane separation to fall thalline; Then protein content is concentrated to about 5% with nanofiltration; Be heated to 80 degree, keep 20 minutes, be cooled to 20 degree, centrifugation impurity elimination, rotating speed 10000rpm, 30min; Add 10 times amount PH5 hydrochloric acid soln dilutions, make protein content about 0.5%, cross DEAE and G50 gel column; Nanofiltration proteins concentrate content is about 5%, and freeze-drying, product purity is 95%, and purified product is compared with the substratum quality of giving money as a gift, and the productive rate of training and peptide is about 0.2%

Productive rate={ (fermented liquid expression product total mass 1 × nanofiltration concentration yield × impurity elimination yield × gel separation yield × concentration yield × freeze-drying yield)/give money as a gift substratum total mass } × 100%.

Step 5. is mixed with reserve liquid

The purified of upper step freeze-drying, uses 0.1N dissolving with hydrochloric acid, makes training and the peptide of 20mg/ml, saves backup.

Embodiment 2. is trained and peptide suppresses hepatitis B replication activity test

Step 1. drug treating

Get one bottle, HepG2.2.15 cell, be prepared into single cell suspension with after 0.25% trysinization, after counting, adjustment cell concn is 5 × 10 ⁴cell/ml, 1ml/ hole adds in 24 well culture plates, puts 37 DEG C, 5%CO ₂overnight incubation in cell culture incubator.Supernatant is abandoned in suction in second day, adds the DMEM substratum containing different pharmaceutical concentration (50,25,12.5,6.25,3.13 and 0 μMs), often organizes 4 multiple holes.Change the cell culture fluid of same drug level after each effect 4d, and in the 4th and 8 days, collect each hole supernatant in 1.5ml centrifuge tube ,-20 DEG C frozen for subsequent use.

The detection of HBsAg and E antigen in step 2. cells and supernatant

Adopt ELISA method to detect HBsAg and HBeAg, concrete operations are carried out according to the operation instruction of test kit.After color reaction terminates, detect each hole OD value by microplate reader at 450nm place.

Step 3. data processing

Data are expressed as means ± SD.

IC (Inhibition rate, inhibiting rate)=(1-OD _{drug treating group}/ OD _{negative control group}) × 100%

Step 4. experimental result, to the restraining effect of hepatitis B replication

HepG2.2.15 cell, after 24 orifice plate overnight incubation, adds the medicine of different weaker concn, sets cell controls group and positive controls simultaneously.After cultivation 4 and 8d, difference collecting cell supernatant, adopts ELISA method to detect the expression of HBsAg and HBeAg.

Tested material training and peptide and positive drug 3TC make the HBsAg secretion level decline in various degree in nutrient solution, have the restraining effect of obvious dosage correlation in the concentration range of 3.13 ~ 50 μ g.The OD value of blank group compares with the same period, and training and each concentration group of peptide obviously decline (p<0.05 or 0.01) (the results are shown in Table 1) at 4d and 8d cell conditioned medium HBsAg content.Training and peptide are approximately 10.9 ~ 20 μ g/ml to the half-inhibition concentration IC50 that cell conditioned medium HBsAg secretes.

Table 1 training and peptide are to the restraining effect of HepG2.2.15 emiocytosis HBsAg

Compare with blank group, ^*p<0.05, ^*p<0.01; Compare with the positive controls under same concentration, ^#p<0.05, ^##p<0.01.

Compared with blank group, training and peptide supernatant HBeAg content when 4d slightly decline.When 8d, training and peptide to the activity inhibition of emiocytosis HBeAg the most obviously, and have certain dose-effect relationship (the results are shown in Table 2).

Table 2 training and peptide process HepG2.2.15 cell 14d are to the inhibiting rate of supernatant HBeAg

Tested material training and peptide and positive drug 3TC have inhibition in various degree to the secretion level of HBsAg and HBeAg in nutrient solution, train and in the concentration range of 3.13 ~ 50 μ g, present the relevant restraining effect of certain dosage with peptide.

30 days feeding studys of embodiment 3. training and peptide piglet

The selection of step 1. piglet: the piglet choosing the child care stage, test group and each 30 of control group

Step 2. test training and peptide quality and standard: non-purifying is trained and peptide crude product, and net content 1 g/kg, containing dead thalline and indigested substratum.

The feeding conventional baby pig feedstuff of step 3. feeding patterns test group, adds training and the peptide crude product of 0.5%; The feeding conventional baby pig feedstuff of control group.

Step 4. observation index: the food consumption of piglet, piglet starting weight, the diarrhoea situation of piglet, surviving rate, weightening finish, hair color, the length of one's sleep, feed record.

Step 5. test-results: the test group piglet of adding training and the raising of peptide crude product, hair color, body weight, body weight, to search for food and body weight is obviously better than control group.In table 3

30 days feeding studys of table 3 training and peptide piglet

Claims (10)

1. training and peptide, is characterized in that having the aminoacid sequence shown in Seq ID No.1.

2. with the aminoacid sequence that Seq ID No.1 is core sequence polypeptide, and Seq ID No.4, Seq ID No.5, Seq ID No.6, the artificial sequence amino acid shown in Seq ID No.7.

3. the gene of coding peptide according to claim 1.

4. gene according to claim 3, its nucleotide sequence is as shown in Seq ID No.2.

5. the expression vector containing the gene described in claim 3 or 4.

6. expression vector according to claim 5, its skeleton carrier is pKLAC1 or pKLAC2.

7. the aminoacid sequence polypeptide as shown in Seq ID No.1, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, SeqID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11 adopts the K.1actis YCT799 preparation of yeast as described in this patent, and similar yeast K.lactis YCT284, YCT389, YCT390, YCT569, YCT598, prepared by yeast saccharomyces cerevisiae, bread yeast, Kluyveromyces lactis and cereuisiae fermentum.

8. the preparation method of the aminoacid sequence polypeptide as shown in Seq ID No.1, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, SeqID No.8, Seq ID No.9, Seq TD No.10, Seq ID No.11, is characterized in that being that the expression vector of pKLAC1 or pKLAC2 is transferred in yeast cell according to claim 7 and carries out fermentation culture by skeleton carrier.

9. the aminoacid sequence polypeptide as shown in Seq ID No.1, Seq ID No.3, Seq ID No.4, Seq ID No.5, Seq ID No.6, Seq ID No.7, SeqID No.8, Seq ID No.9, Seq ID No.10, Seq ID No.11 is preparing the application of hepatitis B medicine in medicine, healthy products, fodder additives, antiviral, immunostimulant product, sleep improvement product, functional food, fodder additives, veterinary drug, growth stimulant.

10. application according to claim 9, described medicine and product refer to oral pharmaceutical, injectable drug, solid and liquid product.