CN106148299A - Protein transduction peptide-PON1 fusion protein and its production and use - Google Patents

Abstract

The present invention relates to protein transduction peptide-PON1 fusion protein and its production and use.Specifically, the present invention relates to mammalian cell and the silkworm expression method of protein transduction peptide PON1 (P11-PON1) fusion protein, this fusion protein has mucosa absorption and enters activity that is internal and that can pass through blood brain barrier.This fusion protein has therapeutical effect to diseases such as organophosphate poisoning and tumor, atherosclerosis, coronary heart disease, type 2 diabetes mellitus.

Description

Protein transduction peptide-PON1 fusion protein and its production and use

Technical field

The present invention relates to the fusion protein of PON1, particularly to protein transduction peptide with The fusion protein of PON1 and the preparation of described albumen and application.

Technical background

PON 1 (paraoxonase 1, PON1) is by liver synthesis secretion Molecular weight is the glycoprotein of 45kDa, containing 355 aminoacid, be primarily present in blood, kidney, In the histoorgan such as spleen, brain.PON1 is a class calcium ion dependency high density lipoprotein phase Close lipase, with hydrolyse phosphate esters key, organic phosphorus compound can be had stronger Detoxication. Further investigation revealed that it plays important work in regulation oxidative stress, lipid metabolism, antiinflammatory With, and with the multiple diseases such as tumor, atherosclerosis, coronary heart disease, type 2 diabetes mellitus Occur closely related.

PON1 is internal trace albumin, extracts this albumen and there is also many problems from serum. In order to study PON1 hydrolysis organophosphorus toxicants and the effect in other diseases, utilize biological work It is the most necessary that Cheng Fangfa expresses PON1.PON1 is glycoprotein, and prokaryotic expression can not be repaiied in glycosylation Decorations, affect protein active.Eukaryotic system expressing protein can be glycosylation modified, feeds so utilizing Breast zooblast and silkworm (being eukaryote), expressing protein can be glycosylation modified, is beneficial to live Property play.Silkworm genetic background is clear, and expressing quantity is high, and edible, some medicinal egg Just can pass through oral absorption by purification in vain.But some albumen digested road proteasome degradation, Reducing its curative effect, this type of protein medicaments being therefore developed for mucosa absorption is the most necessary.

Summary of the invention

In order to provide can the PON1 albumen of mucosa absorption, the present invention provides a kind of peptide Han protein transduction PON1 fusion protein and preparation method thereof.The fusion protein of the present invention can be used for preparing The dosage forms such as buccal tablet, nasal spray, rectum feed for treatment tumor, atherosclerosis, The disease such as coronary heart disease, type 2 diabetes mellitus and in vivo or external degradation organophosphor.

Research finds, has and be referred to as protein transduction district on some protein The small fragment of (protein transduction domain, PTD), is also called Cell-penetrating peptide (cell-penetrating peptides, CPP) can be effectively By biomembrane, enter various types of cell.Exogenous proteins, DNA or compound After thing is connected with PTD, it is possible to enter cell and pass through blood brain barrier.Therefore, PTD Transporting protein, DNA or complex, and and then infect in treatment virus, special The property aspect such as killing tumor cell and neurodegenerative diseases has huge potentiality.

Human immunodeficiency virus-1 (human immunodeficiency virus-1, HIV-1) antisense activated transcription (trans-activator transcription, TAT) Albumen PTD (TAT-PTD) is a kind of source of current PTD.In TAT 11 Aminoacid (47-57 amino acids, P11) is the peptide with protein transduction Section, its sequence is YGRKKRRQRRR (SEQ ID NO.1).TAT-PTD can be by therewith The polypeptide being connected and full-length proteins are transduceed in several minutes and are entered cell, and can lead to Cross blood circulation transport to cerebral tissue, thus may span across blood brain barrier enter neuron or In glial cell.

Obtain can the melting of mucosa absorption by being merged by PON1 with P11 for the present inventor Hop protein.It is demonstrated experimentally that the fusion protein of the present invention can pass through mucosa absorption, noinvasive Property penetrate mucosa enter internal, and then can be used for treat tumor, atherosclerosis, The disease such as coronary heart disease, type 2 diabetes mellitus and in vivo or external degradation organophosphor.

Specifically, the invention provides following various aspects.

One aspect of the present invention provides the fusion protein of P11 Yu PON1.

Optionally, PON1 can be full-length proteins or the activity form for its truncate.

More specifically, the fusion protein of the present invention can have a following sequence:

YGRKKRRQRRRMAKLIALTLLGMGLALFRNHQSSYQTRLNALREVQPVELPNCNLVKGI ETGSEDL/MEILPNGLAFISSGLKYPGIKSFNPNSPGKILLMDLNEEDPTVLELGITGSKFDVSSFN PHGISTFTDEDNAMYLLVVNHPDAKSTVELFKFQEEEKSLLHLKTIRHKLLPNLNDIVAVGPEHFY GTNDHYFLDPYLQ/RSWEMYLGLAWSYVVYYSPSEVRVVAEGFDFANGINISPDGKYVYIAELL AHKIHVYEKHANWTLTPLKSLDFNTLVDNISVDPETGDLWVGCHPNGMKIFFYDSENPPASEVL RIQNILTEEPKVTQVYAENGTVLQGSTVASVYKGKLLIGTVFHKALYCEL (SEQ ID NO:2).

So, one aspect of the present invention provides a kind of fusion protein, and it comprises selected from such as Under sequence:

A) aminoacid sequence shown in SEQ ID NO.2；Or

B) aminoacid sequence in (a) is through replacing, lack, inserting or add one Individual or several aminoacid and retain replaces, lack, insert or adds before active by (a) Derivative aminoacid sequence.

Preferably, the fusion protein of the present invention is:

A) aminoacid sequence shown in SEQ ID NO.2；With

B) aminoacid sequence in (a) is through replacing, lack, inserting or add one Individual or several aminoacid and retain replaces, lack, insert or adds before active by (a) Derivative aminoacid sequence.

Of the present invention replace in aminoacid sequence, lack, insert or add 1 Individual or several aminoacid, refers to any and 1 or several aminoacid in the sequence Replace, lack, insert or add 1 on position in sequence or several aminoacid is residual Base (such as 2,3,4,5,6,7,8 or 9), And in replacing, lack, insert and adding 2 kinds or two or more can occur simultaneously.

Of the present invention replace in aminoacid sequence, lack, insert or add 1 Individual or several aminoacid (can divide by using " Molecular Cloning 3 " Son clone 3) and " Current Protocols in Molecular Biology " The site-directed mutagenesis that (modern molecular biology rule of operation) etc. are recorded obtains.

For replacement of the present invention, preferably within following each group, carry out aminoacid The mutual replacement of residue:

1: leucine, valine, alanine, methionine, serine, glycine；

2: aspartic acid, glutamic acid；

3: agedoite, glutamine；

4: lysine, arginine；

5: proline, hydroxyproline；

6: serine, threonine；With

7: phenylalanine, tyrosine.

For conservative replacement specifically described herein, lack, insert or add front activity, The protein after replacing, lack, insert or adding or aminoacid sequence can be represented Activity be more than 10% before replacing, lack, insert or adding, more than 20%, More than 40%, more than 60%, more than 80%, more than 90%, more than 95%, 96% Above, more than 97%, more than 98%, more than 99% or 100% or higher.

Above-mentioned disappearance, the number of the amino acid residue replacing, insert and/or adding, The least number.And this proteinoid can also be: protein, its tool Have the aminoacid sequence with serial number 5 have about 60% or above, about 70% or above, 71% or above, 72% or above, 73% or above, 74% or above, 75% or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1% or with Upper, 99.2% or above, 99.3% or above, 99.4% or above, 99.5% or with Upper, 99.6% or above, 99.7% or above, 99.8% or above or 99.9% or The aminoacid sequence of above homogeneity and have what SEQ ID NO:2 was had Non-invasive high-penetrability PON1 activity.The numerical value of above-mentioned homology is the biggest Numerical value.

The fusion protein of the present invention removes by gene engineering method (such as following embodiment institute State) obtain outside, it is also possible to by Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc The chemical synthesis manufactures such as method (tertbutyloxycarbonyl method), or by peptide synthesizer Learn synthesis.

Another aspect of the present invention additionally provides the core of the fusion protein of code book invention Nucleotide sequence.

More specifically, the nucleotide sequence of the fusion protein of code book invention is as follows:

The coding nucleotide of the present invention can have a following sequence: TATGGTCGTAAAAAACGTCGCCAGCGTCGCCGTAtggcgaagctgattgcgctcaccctcttggggatggga ctggcactcttcaggaaccaccagtcttcttaccaaacacgacttaatgctctccgagaggtacaaccc gtagaacttcctaactgtaatttagttaaaggaatcgaaactggctctgaagacttg/atggagatact gcctaatggactggctttcattagctctggattaaagtatcctggaataaagagcttcaaccccaacag tcctggaaaaatacttctgatggacctgaatgaagaagatccaacagtgttggaattggggatcactgg aagtaaatttgatgtatcttcatttaaccctcatgggattagcacattcacagatgaagataatgccat gtacctcctggtggtgaaccatccagatgccaagtccacagtggagttgtttaaatttcaagaagaaga aaaatcgcttttgcatctaaaaaccatcagacataaacttctgcctaatttgaatgatattgttgctgt gggacctgagcacttttatggcacaaatgatcactattttcttgacccctacttacaa/cgatcctggg agatgtatttgggtttagcgtggtcgtatgttgtctactatagtccaagtgaagttcgagtggtggcag aaggatttgattttgctaatggaatcaacatttcacccgatggcaagtatgtctatatagctgagttgc tggctcataagattcatgtgtatgaaaagcatgctaattggactttaactccattgaagtcccttgact ttaataccctcgtggataacatatctgtggatcctgagacaggagacctttgggttggatgccatccca atggcatgaaaatcttcttctatgactcagagaatcctcctgcatcagaggtgcttcgaatccagaaca ttctaacagaagaacctaaagtgacacaggtttatgcagaaaatggcacagtgttgcaaggcagtacag ttgcctctgtgtacaaagggaaactgctgattggcacagtgtttcacaaagctctttactgtgagctct Aa (SEQ ID NO:3).

So, the nucleotide sequence of the fusion protein of code book invention comprises:

A) nucleotide sequence shown in SEQ ID NO.3；

B) under stringent condition nucleotide sequence with SEQ ID NO.3 carry out hybridizing, And the polynucleotide of the fusion protein of code book invention；Or

C) above-mentioned complementary series a) or b).

It is further preferred that the nucleotides sequence of the fusion protein of code book invention is classified as:

A) nucleotide sequence shown in SEQ ID NO.3；

B) under stringent condition with carried out miscellaneous by the nucleotide sequence of SEQ ID NO.3 Hand over and encode the polynucleotide of the aminoacid sequence with fusion protein of the present invention activity；

C) above-mentioned complementary series a) or b).

Preferably, the nucleotide sequence of the present invention can comprise restriction enzyme site, such as NCOI/XhoI, exemplarily, NCOI+P11+PON1+XhoI sequence is as follows:

CCATGGGCTATGGTCGTAAAAAACGTCGCCAGCGTCGCCGTAtggcgaagctgattgcgctcaccctctt ggggatgggactggcactcttcaggaaccaccagtcttcttaccaaacacgacttaatgctctccgaga ggtacaacccgtagaacttcctaactgtaatttagttaaaggaatcgaaactggctctgaagacttg/a tggagatactgcctaatggactggctttcattagctctggattaaagtatcctggaataaagagcttca accccaacagtcctggaaaaatacttctgatggacctgaatgaagaagatccaacagtgttggaattgg ggatcactggaagtaaatttgatgtatcttcatttaaccctcatgggattagcacattcacagatgaag ataatgccatgtacctcctggtggtgaaccatccagatgccaagtccacagtggagttgtttaaatttc aagaagaagaaaaatcgcttttgcatctaaaaaccatcagacataaacttctgcctaatttgaatgata ttgttgctgtgggacctgagcacttttatggcacaaatgatcactattttcttgacccctacttacaa/ cgatcctgggagatgtatttgggtttagcgtggtcgtatgttgtctactatagtccaagtgaagttcga gtggtggcagaaggatttgattttgctaatggaatcaacatttcacccgatggcaagtatgtctatata gctgagttgctggctcataagattcatgtgtatgaaaagcatgctaattggactttaactccattgaag tcccttgactttaataccctcgtggataacatatctgtggatcctgagacaggagacctttgggttgga tgccatcccaatggcatgaaaatcttcttctatgactcagagaatcctcctgcatcagaggtgcttcga atccagaacattctaacagaagaacctaaagtgacacaggtttatgcagaaaatggcacagtgttgcaa ggcagtacagttgcctctgtgtacaaagggaaactgctgattggcacagtgtttcacaaagctctttac tgtgagctctaaCTCGAG(SEQ ID NO:4).

" stringent condition " as herein described, can be low stringent condition, in tight bar Any one in part, high stringent condition, the highest stringent condition.Exemplarily, " low stringent condition " can be 30 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, The condition of 52% Methanamide；" middle stringent condition " can be 40 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, the condition of 52% Methanamide；" high stringent condition " Can be 50 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, the bar of 52% Methanamide Part.Skilled artisan would appreciate that temperature more Gao Yueneng obtains high homology many Nucleotide.It addition, those skilled in the art can select the stringency of impact hybridization Temperature, concentration and probe concentration, probe length, ionic strength, time, salinity etc. are multiple The synthesis result that factor is formed realizes corresponding stringency.

The most interfertile polynucleotide can also be, by FASTA, BLAST When calculating Deng the default parameters of homology search software default, with coding The polynucleotide of serial number 6 have about 60% or above, about 70% or above, 71% Or above, 72% or above, 73% or above, 74% or above, 75% or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or with Upper, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above or 99.9% or more than The polynucleotide of homogeneity.

Aminoacid sequence, the homogeneity of nucleotide sequence, it is possible to use Karlin and The algorithmic rule BLAST of Altschul (Proc.Natl.Acad.Sci.USA 87: 2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993) come Determine.Program BLASTN based on BLAST algorithm rule, BLASTX have been developed that (Altschul SF,et al:J Mol Biol 215:403,1990).Use BLASTN When analyzing base sequence, as made parameter be score=100, wordlength=12； In addition use BLASTX analysis of amino acid sequence time, as make parameter be score=50, Wordlength=3；When using BLAST and Gapped blast program, use each The system of program can set default parameter value.

Present invention also offers the carrier of the nucleotide sequence comprising the present invention, such as matter Grain carrier.The micro-of nucleotide of the present invention is imported it addition, present invention also offers Biology, such as virus.

Present invention also offers the method preparing fusion protein of the present invention, its bag Include the nucleotide sequence making the present invention to express in silkworm.

Another aspect of the invention provides a kind of pharmaceutical composition or pharmaceutical preparation, its Comprise fusion protein or its nucleotide sequence of the present invention.Medicine group for the present invention Compound or pharmaceutical preparation, its can be used for treat tumor, atherosclerosis, coronary heart disease, Diseases such as type 2 diabetes mellitus and in vivo or external degradation organophosphor.

It is yet another aspect of the present invention to provide fusion protein and the nucleotide thereof of the present invention Sequence is used for treating tumor, atherosclerosis, coronary heart disease, type 2 diabetes mellitus etc. in preparation In the medicine of disease or for the purposes in vivo or in the medicine of external degradation organophosphor.

For the pharmaceutical composition of the present invention, it can pass through buccal tablet, nasal spray, straight The dosage forms such as enteral administration are administered.

Accompanying drawing explanation

Fig. 1: silkworm expression vector construction and infection flow process.

The SDS-PAGE figure of Fig. 2: P11-PON1 fusion protein, wherein:

M: albumen Marker；1,2: two batches of protein samples expressed.

The Westblott ing of Fig. 3: P11-PON1 fusion protein identifies, wherein:

1,2: two batches of protein samples expressed.

Fig. 4: the P11-PON1 fusion protein biological activity to mice.

Fig. 5: the P11-PON1 fusion protein biological activity to Brachydanio rerio.

Below will be by illustrating in greater detail the present invention by following example.Following reality Execute example to be merely illustrative, it is to be understood that the present invention is not limited by following embodiment.

Embodiment 1. silkworm expression vector construction and silkworm expression

Synthetic P11-PON1 fusion gene NCOI+PTD+PON1+XhoI sequence is as follows:

CCATGGGCTATGGTCGTAAAAAACGTCGCCAGCGTCGCCGTAtggcgaagctgattgcgctcaccctctt ggggatgggactggcactcttcaggaaccaccagtcttcttaccaaacacgacttaatgctctccgaga ggtacaacccgtagaacttcctaactgtaatttagttaaaggaatcgaaactggctctgaagacttgga gatactgcctaatggactggctttcattagctctggattaaagtatcctggaataaagagcttcaaccc caacagtcctggaaaaatacttctgatggacctgaatgaagaagatccaacagtgttggaattggggat cactggaagtaaatttgatgtatcttcatttaaccctcatgggattagcacattcacagatgaagataa tgccatgtacctcctggtggtgaaccatccagatgccaagtccacagtggagttgtttaaatttcaaga agaagaaaaatcgcttttgcatctaaaaaccatcagacataaacttctgcctaatttgaatgatattgt tgctgtgggacctgagcacttttatggcacaaatgatcactattttcttgacccctacttacaatcctg ggagatgtatttgggtttagcgtggtcgtatgttgtctactatagtccaagtgaagttcgagtggtggc agaaggatttgattttgctaatggaatcaacatttcacccgatggcaagtatgtctatatagctgagtt gctggctcataagattcatgtgtatgaaaagcatgctaattggactttaactccattgaagtcccttga ctttaataccctcgtggataacatatctgtggatcctgagacaggagacctttgggttggatgccatcc caatggcatgaaaatcttcttctatgactcagagaatcctcctgcatcagaggtgcttcgaatccagaa cattctaacagaagaacctaaagtgacacaggtttatgcagaaaatggcacagtgttgcaaggcagtac agttgcctctgtgtacaaagggaaactgctgattggcacagtgtttcacaaagctctttactgtgagct ctaaCTCGAG(SEQ ID NO:5).

Aminoacid sequence coded by above-mentioned nucleotide sequence is as follows:

YGRKKRRQRRRMAKLIALTLLGMGLALFRNHQSSYQTRLNALREVQPVELPNCNLVKGIETGSE DLEILPNGLAFISSGLKYPGIKSFNPNSPGKILLMDLNEEDPTVLELGITGSKFDVSSFNPHGISTFT DEDNAMYLLVVNHPDAKSTVELFKFQEEEKSLLHLKTIRHKLLPNLNDIVAVGPEHFYGTNDHYF LDPYLQSWEMYLGLAWSYVVYYSPSEVRVVAEGFDFANGINISPDGKYVYIAELLAHKIHVYEKH ANWTLTPLKSLDFNTLVDNISVDPETGDLWVGCHPNGMKIFFYDSENPPASEVLRIQNILTEEPK VTQVYAENGTVLQGSTVASVYKGKLLIGTVFHKALYCEL (SEQ ID NO:6).

The two ends of nucleotide sequence based on synthesis add NcoI and XhoI restriction enzyme site, Utilize NcoI and XhoI double digestion above-mentioned P11-PON1 fusion gene and pFastBac respectively HTb carrier (Invtrogen), reclaims P11-PON1 and carrier segments, and T4DNA is even Connect enzyme to connect, then will connect product and convert DH5_ɑCompetent cell, picking list bacterium colony, Obtain recombinant vector pFas tBac5B-P11-PON1.Then convert with this vector plasmid DH10BmBac competent cell (Invtrogen), concussion training on LB fluid medium Support 4h, then take part of dilution bacterium solution coated plate, containing tetracycline and kanamycin mycin With grow 48h on the LB of X-gal.Color according to bacterium colony is screened, picking white macula Carrying out single bacterial plaque to cultivate, extracting obtains BmBacmid gene of recombinating.It is transfected into silkworm subsequently Cultivate cell and obtain recombinant virus by conventional method.Then this recombinant virus infection is used Silkworm 5 instar larvae, i.e. has P11-PON1 expressing fusion protein after infecting 96 hours, receives Collection polypide lyophilizing is pulverized, and-80 DEG C save backup (see Fig. 1).

The qualification of embodiment 2 P11-PON1 fusion protein

Take 1 gram of polypide dry powder, be dissolved in 20mlPBS solution, 4 DEG C of ultrasonication 20min, Under 12000r/min, centrifugal 10min, collects supernatant, utilizes BCA quantification of protein reagent Box measures silkworm Tot Prot；Take 10 μ l supernatants and carry out SDS-PAGE electrophoresis, configure two Block 12%SDS-PAGE gel, after electrophoresis terminates, one piece is placed in coomassie brilliant blue staining 1h, Glacial acetic acid decolours overnight, utilizes Gel-pro gel imaging system take pictures and scan calculating P11-PON1 fusion protein accounts for total protein ratio, final calculating P11-PON1 fusion protein Net content；Another block glue is identified for western blot, after transferring film 37 DEG C, 5% defat 2h closed by milk, and one anti-(rabbit anti-human PON1 polyclonal antibody 1:1000 dilution) 4 DEG C incubates Educating overnight, two anti-(1:2000 dilution) 37 DEG C hatches 2h, utilizes enhancement mode HRP-DAB Substrate colour reagent box directly develops the color on pvdf membrane, utilizes Gel-pro after colour developing Gel imaging system is taken pictures.P11-PON1 fusion protein silkworm expression molecular weight 45kDa is left Right (Fig. 2), accounts for the 5-10% of silkworm total protein, and western blot is identified the most correct (Fig. 3).

The biological activity of embodiment 3 P11-PON1 fusion protein

The 3.1 P11-PON1 fusion protein biological activity to mice

Kunming mice male and female half and half, body weight 25 ± 3 grams, before experiment, fasting 12 hours, makes The emptying as far as possible of harmonization of the stomach intestinal, freely drinks water.Experiment component is gavage group and rectally group, Matched group lumbar injection dichlorvos 20mg/kg body weight.Experiment is repeated 3 times, often group 6, Male and female half and half.Every mice of gavage group directly gives to express supernatant by gastric perfusion needle (P11-PON1 fusion protein and PON1 albumen (PON1 albumen net content 1mg)；Rectum Administration group, first stimulates mice anus by gastric perfusion needle so that it is emptying internal rectum feces as far as possible, Probe into about 1.5cm by gastric perfusion needle again, be slowly injected into, stop about 1min and slowly remove pin To prevent medicinal liquid from flowing out.After being administered 1-5 hour, experimental group and matched group press 20mg/kg Body weight lumbar injection dichlorvos solution, observes mice state, records survival rate.Gavage is given Medicine does not improve mouse survival rate；And rectally after 1-5 hour again lumbar injection enemy enemy Fear, P11-PON1 fusion protein group mouse survival rate pole significantly improves (P < 0.01)；Table Bright P11-PON1 fusion protein can be degraded by gastrointestinal tract enzyme, and loses activity, but can lead to Cross mucosa and enter internal, and organophosphorus toxicants is had resistant function；And the PON1 without P11 Albumen can not enter internal by mucosa, does not has resistant function (see Fig. 4) to organophosphorus toxicants.

The 3.2 P11-PON1 fusion protein biological activity to Brachydanio rerio

Experiment packet: experimental group and matched group.Experimental group, P11-PON1 fusion protein and PON1 albumen (with the net content of PON1 albumen) final concentration is respectively 1,2.5,5,10, 20mg/L, puts into state normal adult Brachydanio rerio, often group 8, male female half and half, 1-5 Add the final concentration of 50mg/L of dichlorvos solution after hour, fully mix；Matched group, will State normal adult Brachydanio rerio is put in 1L cultivation water, often group 8, and male female half and half, with Experimental group is simultaneously introduced the final concentration of 50mg/L of dichlorvos solution, fully mixes；Experiment periods Between pull dead fish in time out, observe 24 hours, record experiment the poisoning symptom of fish, death toll Amount, evaluation fish is the most dead the most movable using the gill and has minimal irritation reactionless as depending on According to, if the gill stop motion of fish in Shi Yan, touch afterbody with Glass rod, reactionless, recognize For death.Calculate each group of survival rate.Experimental result, each group of P11-PON1 fusion protein, 20mg/L group 24h survival rate 62.5%, 10mg/L group survival rate 62.5%, 5mg/L group Survival rate 50%, compared with matched group, survival rate pole significantly improves (P < 0.01)；2.5mg/L Group survival rate 41.7% significant difference (P < 0.05) compared with matched group；1mg/L group is deposited Motility rate 16.7%, compared with matched group, not statistically significant.Show that P11-PON1 merges Albumen and can have opposing to organophosphorus toxicants by the cheek with mucocutaneous be absorbed into internal Effect；And PON1 protein groups is respectively organized survival rate and is not the most significantly improved, to organophosphorus toxicants There is no resistant function (see Fig. 5).

Although with above embodiments describing the present invention, it should be appreciated that not On the premise of deviating from the spirit of the present invention, the present invention can further be modified and be become Dynamic, and these modify and within variation belongs to protection scope of the present invention.

Claims (16)

1. fusion protein, it is following form: the albumen of SEQ ID NO:1 turns Lead peptide+PON1.

Fusion protein the most according to claim 1, wherein PON1 is Total length or activated truncate form.

Fusion protein the most according to claim 1, wherein said fusion protein For SEQ ID NO:2.

Fusion protein the most according to claim 3, wherein said fusion protein For SEQ ID NO:6.

5. according to the fusion protein according to any one of claim 1-4, Qi Zhongsuo State fusion protein to produce by expressing in silkworm.

6. according to the fusion protein according to any one of claim 1-4, Qi Zhongsuo State fusion protein to be contained in silkworm or in silkworm powder.

7. the nucleotide of coding fusion protein any one of claim 1-6.

Nucleotide the most according to claim 7, wherein said nucleotide bag Sequence containing SEQ ID NO:3 or 4.

Nucleotide the most according to claim 8, its sequence is SEQ ID NO: 5。

10. expression vector, it comprises the nucleotide any one of claim 7-9.

11. carriers according to claim 10, it is plasmid vector or viral vector.

12. methods preparing fusion protein, it includes utilizing silkworm expression system expression Fusion protein any one of claim 1-6.

13. pharmaceutical compositions, it comprises the fusion any one of claim 1-6 Albumen or according to the fusion protein prepared by claim 12.

14. compositionss according to claim 13, it is buccal tablet, nose spray Agent or forms for rectal administration.

Fusion protein any one of 15. claim 1-6 or according to claim The fusion protein prepared by 12 purposes in preparing medicine, wherein said medicine For treating organophosphorus toxicants poisoning, tumor, atherosclerosis, coronary heart disease or 2 Patients with type Ⅰ DM.

16. purposes according to claim 15, wherein said medicine be buccal tablet, Nasal spray, the dosage form of rectally.