CN1482242A - A anti-enveloped virus recombinant polypeptide and its preparation method - Google Patents
A anti-enveloped virus recombinant polypeptide and its preparation method Download PDFInfo
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Abstract
The present invention relates to one kind of enveloped virus resisting polypeptide and its encoding nucleotide and amino acid sequence. Colicin capable of forming ion channel or its aquatic porous channel structural domain is made to combine with polypeptide capable of being combined specifically with target virus envelope antigen to form engineering polypeptide. Inside the engineering polypeptide, ScFv antigen combining area capable of being combined specifically with target virus envelope antigen induces the colicin water soluble porous channel structural domain to near target virus envelope, the colicin water soluble porous channel structural domain then forms the ion channel on the envelope to destroy the completeness of the envelope to leak or expose virus RNA and kill virosome. The antiviral engineering polypeptide kills selected virosome without injuring host cell, being superior to traditional antiviral medicines.
Description
Technical field
The present invention relates to a kind of antiviral polypeptide of reorganization, relate in particular to the polypeptide of anti-enveloped virus, the Nucleotide of this polypeptide of encoding and aminoacid sequence.
The invention still further relates to the preparation method of reorganization antiviral polypeptide.
Background technology
Virus infection has become human life and healthy main threat, along with global biosphere is day by day destroyed by human industrialization behavior, virus disease, be becoming increasingly rampant as viral hepatitis, influenza, pneumonia, encephalitis, virogenetic tumour, AIDS etc., because the gene of virus is easy to undergo mutation, virogenetic damage often has the function error of host immune system to participate in, its mechanism is still among exploring, so people are making great efforts to attempt to scheme to prepare real effectively antiviral always.
Antiviral therapy is many at present sets about from following several respects: 1, suppress viral nucleic acid, and 2, suppress virus protease 3, utilize immune factor to kill and wound, such as Interferon, rabbit, 4, utilize virus vaccines immunity etc.
Yet in actual applications, virus utilizes its sudden change characteristics often can allow above-mentioned treatment means lose efficacy soon.Such as the anti-hepatic-B virus medicine Laminvudine that came into operation before the several years, efficiently when just bringing into use severally reach 90%, yet only pass through the clinical application of several years, because it is the gene of hepatitis B virus undergos mutation rapidly, efficient from about 90% reducing to and have only about 10% originally.Therefore must seek other more efficiently antiviral by way of.
Based on above-mentioned present situation, the patent inventor thinks that the structure that select virus to be difficult for undergoing mutation kills and wounds virus as pharmaceutically-active target spot, could fundamentally avoid existing antiviral therapy means so easily by virus mutation institute destructive deadly defect.
Patent inventor's imagination, at tunicary virosome,, albumen in the coating and Yeast Nucleic Acid are leaked or expose and cause virus dead if can destroy the integrity of its coating, just might stop the infection and the repeated infection of virus, thereby reach the purpose of treatment.And peplos is made of bimolecular lipid membrane, is the biological structure of quite stable, and the probability of undergoing mutation is very low for Yeast Nucleic Acid and protein.And at occurring in nature, there is bacteriotoxin quite a lot to come killing bacteria with the integrity of the bimolecular lipid membrane that destroys host cell membrane, use for reference the antibiosis mechanism that more than one hundred million years struggle for existences of this process preferably come out so why not and design the novel antiviral medicine?
Nineteen fifty-two Jacob finds some bacillus of intestinal bacteria that colicin can kill other strain system specifically and relevant strain system, as Shigella Sammei (Jacob et al, Sur labiosynthese d ' unecolicine et son mode d ' action, Annals of the Pasteur Institute.83:295-315 (1952)).Finkelstern in 1978 etc. have found that the colicin that can form ionic channel can form the voltage-dependent ionic channel on artificial bimolecular lipid membrane, thereby at the antibacterial mechanisms that has fundamentally disclosed this bacterioid toxin (Schein et al .Colicin K acts by foming voltagedependent channels in phospholipid bilayer membranes.Nature 276:159-163 (1973)).Qiu in 1996 and Finkelstein etc. have disclosed ionic channel that colicin Ia forms on artificial bimolecular lipid membrane open and stride film three-dimensional arrangement (Qiu et al. when closing, Majortransmenbrane movement associated with colicin Ia chammel gating.J.Gen.Physiology.107:313-328 (1996)), established theoretical basis on molecular level, designing and prepare novel antibiotic.But its shortcoming is to act on Gram-negative bacillus such as intestinal bacteria, if can utilize can with antigen-specific bonded biomacromolecule on the peplos as inductor, colicin or its water-based pore passage structure be induced to form ionic channel near the specific peplos and kill and wound this virus, then can enlarge the range of application of colicin greatly.
Enveloped virus is all having highly specific antigen or acceptor between kind on its coating.Such as hepatitis B virus, the HBsAg antigen and the acceptor (medical microbiology, national high medical college teaching material, People's Press's calendar year 2001 version, 292 pages) of liver cell acceptor just arranged on its coating.Therefore, if can utilize can with the polypeptide of envelope antigen specific combination, such as the antigenic single-chain antibody scFv of special resisting HBV virus HBsAg or antigen binding domain wherein as inductor, some can be formed the bacteriotoxin of ionic channel, a kind of toxin protein of intestinal bacteria (Escherichia coli) excretory for example---colicin (colicin) or its water-based pore passage structure territory (pore forming region) is induced to and forms ionic channel on the specific target peplos and kill and wound this virosome, should be a kind of ideal antiviral exploitation direction.
Summary of the invention
One object of the present invention is to provide a kind of novel reorganization antiviral polypeptide, and it acts on the coating of virus, reaches the effect that kills and wounds virosome thereby form ionic channel.
Another object of the present invention is to provide the nucleotide sequence of this reorganization antiviral polypeptide of coding.
A further object of the present invention is to provide a recombinant plasmid, and it contains the nucleotide sequence of code book invention reorganization antiviral polypeptide.
A further object of the present invention is to provide the monoamino-acid sequence, its reorganization antiviral polypeptide of the present invention of encoding.
A further object of the present invention is to provide and contains the recombinate pharmaceutical composition of antiviral polypeptide of the present invention.
A further object of the present invention is to provide the preparation method of reorganization antimicrobial polypeptide.
According to an aspect of the present invention, can form the colicin (colicin) of ionic channel or its water-based pore passage structure territory and can become the recombined engineering polypeptide with the polypeptides in combination of target envelope antigen specific combination.In this project polypeptide, can induce the water-soluble pore passage structure of colicin territory to arrive near the target peplos as inductor with the polypeptide of target envelope antigen specific combination, colicin water-based pore passage structure territory forms ionic channel on coating then, destroy the integrity of coating, kill and wound virosome thereby viral ribonucleic acid is leaked or expose.
In a preferred embodiment of the invention, aminoterminal or the carboxyl terminal of special hepatitis B virus resisting (HBV) PreSlscFv antibody gene (GenBank AF427148 login) with the gene in coding colicin or its water-soluble pore passage structure territory is connected, forms the reorganization antiviral polypeptide.In addition, the length of peptide chain that can be by changing recombinant polypeptide and amino-acid residue are formed and are strengthened its antiviral activity and reduce possible toxic side effect, because such composition or change will increase or reduce the quantity of the amino-acid residue of reorganization antiviral polypeptide, therefore the molecular weight of the reorganization antiviral polypeptide that obtains will be 5, change in 000～100,000 the scope.The optional self energy of colicin among the present invention forms colicin E1, Ia, Ib, A, B and N or its water-based pore passage structure territory of ionic channel, in one embodiment of the invention, the anti-HBV PreSl scFv of GenBank AF427148 login is connected to the aminoterminal in the water-soluble pore passage structure of colicin Ia territory, obtains the recombinant polypeptide of molecular weight about 40,000; In another embodiment, the carboxyl terminal with the anti-HBV PreSl scFv of GenBank AF427148 login is connected to the water-soluble pore passage structure of colicin Ia territory obtains the recombinant polypeptide of molecular weight about 40,000; In another embodiment, the aminoterminal with the anti-HBV PreSl scFv of GenBank AF427148 login is connected to colicin Ia obtains the recombinant polypeptide of molecular weight about 90,000; The anti-HBV PreSl scFv of GenBank AF427148 login is connected to the carboxyl terminal of colicin Ia, obtains the recombinant polypeptide of molecular weight about 90,000.
According to a further aspect in the invention, in order further to improve the activity of antiviral polypeptide, reduce its toxic side effect, can select to adopt with peplos on antigen-specific bonded single-chain antibody (scFV) or antigen binding domain wherein be connected with colicin or its water-soluble pore passage structure territory, obtain active higher antiviral polypeptide like this.
In another preferred embodiment of the present invention, HBV envelope antigen HBsAg single-chain antibody (ScFv) or one of them or several antigen binding domain peptide chains are connected with the gene in coding colicin or its water-soluble pore passage structure territory.In a specific embodiment, the aminoterminal of the gene (SEQ ID NO.2) of the G28-V50 among the coding anti-HBsAg single-chain antibody scFv of GenBank AF 236816 login and two sections peptide chains of E216-S228 is connected with the carboxyl terminal of the gene in encode colicin Ia (SEQ ID NO.1) or its water-soluble pore passage structure territory (K544-I622), form the Nucleotide (SEQ ID NO.4) of coding reorganization antiviral polypeptide, its coding is as the aminoacid sequence of SEQ ID NO.5.In addition, the length of peptide chain that also can be by changing recombinant polypeptide and amino-acid residue are formed and are strengthened its antiviral activity and reduce possible toxic side effect, because such composition or change will increase or reduce the quantity of the amino-acid residue of reorganization antiviral polypeptide, therefore the molecular weight of the reorganization antiviral polypeptide that obtains will be 15, change in 000～74,000 the scope.
In the reorganization antiviral polypeptide that makes up, but the polypeptide of the single-chain antibody antigen binding domain of specific combination envelope antigen induces the water-soluble pore passage structure of colicin territory to pass peplos as inductor, colicin water-based pore passage structure territory forms ionic channel on the target peplos then, thereby reaches the purpose of killing and wounding virus.
According to a further aspect in the invention, the plasmid vector that comprises Nucleotide of the present invention as shown in Figure 1 is provided, and this plasmid vector is the nucleotide sequence of the polypeptide of aforesaid specific combination hepatitis B virus (HBV) envelope antigen single-chain antibody antigen binding domain to be inserted through double-stranded oligonucleotide point mutation technology form recombinant plasmid of the present invention on the 626th amino acids of colicin Ia gene.
Among the present invention, make up the original plasmid pSELECT of plasmid vector
TM-1 comes from Promega company, colicin Ia and immunity protein gene have wherein been loaded, polypeptide gene at specific combination hepatitis B virus (HBV) envelope antigen single-chain antibody antigen binding domain has designed four pairs of primers, and its sequence is respectively shown in SEQ ID NO:6～SEQ ID NO:13.Utilize double-stranded oligonucleotide point mutation technology, obtain recombinant plasmid, again the recombinant plasmid that obtains is transfected into the e. coli tg1 engineering bacteria and obtains host cell according to the medicine-chest operation of Strategene company.
In accordance with a further aspect of the present invention, provide and contain the recombinate pharmaceutical composition of antiviral polypeptide of the present invention, can make the pharmaceutical composition that is suitable for clinical use by antiviral polypeptide of the present invention is added pharmaceutically acceptable carrier or vehicle or optional additional components.
According to another aspect of the invention, the preparation method of antiviral polypeptide of the present invention is provided, the gene of the polypeptide of the specific combination of will encoding hepatitis B virus (HBV) envelope antigen single-chain antibody antigen binding domain operationally is connected the gene that obtains coding reorganization antiviral polypeptide with the colicin gene, the gene that obtains imported in the expression system express, separate polypeptide expressed and obtain antiviral polypeptide of the present invention.
Each enveloped virus all has own unique envelope antigen or acceptor, therefore as long as adopt and to be connected with the colicin that can form ionic channel or its water-soluble pore passage structure territory with the polypeptide of its specific combination, just can be combined into a kind of antiviral recombined engineering polypeptide special, that resist this virus.That is to say that of the present invention one big advantage is multiplely can come with the polypeptide of different virus envelope antigen or receptors bind and the antiviral of any enveloped virus of antagonism is formed in colicin or its water-soluble pore passage structure territory by changing.
Another big advantage of the present invention is that the antiviral engineered polypeptide of preparation can directly form ionic channel to destroy the integrity of coating on the virosome coating, this mode of killing the virus replaces the nucleoside medicine of viral DNA precursor with using nucleic acid analog, suppress the virus protease medicine, Interferon, rabbit and Chinese medicine etc. suppress virus drugs, traditional methods such as immunosuppressor and genetically engineered nucleic acid vaccine are compared, what its benefit was its employing is a kind of the simplest mechanism of effectively killing organism that nature preferably comes out through natural selection in 1 years, this mechanism has very high target, so just guaranteed that this antiviral engineered polypeptide only kills selected virosome and do not injure host cell, thereby reduced the toxic side effect of above-mentioned traditional antiviral to greatest extent the host.
Description of drawings
Fig. 1 illustrates the plasmid structure that contains in the anti-HBV single-chain antibody two sections peptide chains and colicin Ia;
Fig. 2 is the electromicroscopic photograph of normal hepatitis B virus (left figure) and the hepatitis B virus (right figure) after antiviral polypeptide of the present invention is handled 1 hour, show after antiviral polypeptide is hatched 1 hour among the figure, the phospho-wolframic acid dyestuff passes coating and enters virus (dyeing deepens in the coating), shows that the integrity of coating is destroyed.(amplifying 50,000 times)
Fig. 3 uses recombinate antibody concentration after the antiviral polypeptide treatment of the present invention for the chimeric in vivo of the mouse people-liver cell model of biochemical/histology hepatitis performance, as can be seen from the figure, to rising in the 4th week, mice serum HBsAg and the anti-HBc antibody of having injected anti-HBV polypeptide all significantly reduce.
Embodiment
Below in conjunction with accompanying drawing, describe the present invention in detail by description to preferred embodiment of the present invention.
[embodiment 1] expresses the structure and the preparation of reorganization antiviral polypeptide of the plasmid of anti-hepatitis B virus polypeptide
Original plasmid is the pSELECT that has loaded colicin Ia and immunity protein gene
TM-1 commercial plasmid (plasmid size 8.3kb, Promega company) (UCSF, P.Gosh gifts).Through double-stranded oligonucleotide point mutation technology (QuickChange
TMKit, Strategeue company) gene with G28-V50 and two sections peptide chains of E216-S228 in the coding anti-HBsAg single-chain antibody of GenBank AF236816 login is inserted on the I626 site of colicin Ia gene through the sudden change of 4 beans-and bullets shooters, the mutant plasmid that has prepared antiviral polypeptide, (as shown in Figure 1).Mutant plasmid is transfected into E.coli TG1 engineering bacteria (AECOM, K.Jakes gifts) lining and prepares polypeptide.
The sudden change program is undertaken by Strategene QuikChange Site-Directed Mutagenesis Kit (Catalog#200518) medicine-chest handbook:
1. prepare the point mutation reactant:
5μl?10X?buffer
2 μ l (10ng) wild-type colicin plasmids
1.25 5 '-3 ' oligonucleotide primer (seeing listed primer sequence) of μ l (125ng) design
1.25 3 '-5 ' oligonucleotide primer (seeing listed primer sequence) of μ l (125ng) design
1μl?dNTP
Distilled water 50 μ l
1μl?pfu
(except that plasmid, primer and distilled water, being reagent that medicine-chest is equipped with)
2. carry out pcr amplification, amplification condition: 95 ℃ of sex change, 35 seconds, anneal 53 ℃, 70 seconds, extend 68 ℃, totally 20 circulations in 17 fens;
3. add (37 ℃, 1 hour) behind the Dpn1 restriction endonuclease 1 μ l digestion mother body D NA chain, get 1 μ l reactant and XL1-Blue competent cell 50 μ l ice and incubate, 42 ℃ of capable thermal shockings 45 seconds, were inserted in the ice 2 minutes again;
4. add NZY and train 220rpm behind the basic 0.5ml, 37 ℃ were shaken bacterium after 1 hour, got 50-100 μ l reactant bed board (LB training base adds 1% agar and adds 50 μ l/ml penbritins, and 37 ℃ are spent the night);
5.18 choose bacterium after hour, follow Qiagene, companies such as Gibco various commercial extract the plasmid medicine-chests extract plasmids all can, order-checking is determined to suddenly change successfully.
6. the TG1 engineering bacteria of plasmid 50ng and preparation being experienced polypeptide cell 50 μ l ice incubated 30 fens, thermal shocking in 90 seconds, get 50-100 μ l reactant adding LB and train basic 0.5ml, 220rpm, 37 ℃ are shaken 37 ℃ on bacterium bed board after 1 hour (LB training base adds 1% agar and adds 50 μ g/ml penbritins), picking colony after 18 hours.
7. increase bacterium in a large number, 8-16 rises FB training base, 250rpm, 37 ℃, 6-8 hour; The centrifugation thalline, 4 ℃, 6000g 20 minutes, gets 4 ℃, 50mM borate buffer (2mM EDTA+2mMDTT) 50-80ml suspension thalline, add behind 0.2M PMSF 250 microlitres (4 ℃ of ultrasonications, 400W, 2 minutes), (4 ℃ of the broken thalline of high speed centrifugation, 75000g, 1.5 hour), get supernatant and add the Vetstrep 5,000,000 deposit D NA of unit, (4 ℃ of high speed centrifugations, 30000g, 10 minutes) after pack the dialysis tubing of molecular weight 15000 in 4 ℃, after 4 liters of dialysed overnight of 50mM borate buffer, (4 ℃ of high speed centrifugations, 30000g, 10 minutes) the back supernatant is splined on the CM ion exchange column, and 4 ℃, 0.3M NaCl+50mM borate buffer wash-out has just obtained the purifying rate and has reached anti-HBV engineered polypeptide more than 90%, about molecular weight 7.4 ten thousand, the crude preparation by using protein content is about about 1～5mg/ml.Through administrations such as abdominal injection and cell culture fluid dosings, do not find that this project polypeptide shows toxic side effect to the human body cell and the experiment mice of vitro culture.
Designed oligonucleotide sequence in the above-mentioned preparation plasmid
For the first time:
5’-3’(SEQ?ID?NO：6)gcg?aat?aag?ttc?tgg?ggt?att?GGA?TTC?ACC?TTC?AGT?GAC?TAC?TAC?ATG?AGCtaa?ata?aaa?tat?aag?aca?ggc
3’-5’(SEQ?ID?NO：7)gcc?tgt?ctt?ata?ttt?tat?tta?GCT?CAT?GTA?GTA?GTC?ACT?GAA?GGT?GAA?TCCaat?acc?cca?gaa?ctt?att?cgc
For the second time:
5’-3’(SEQ?ID?NO：8)acc?ttc?agt?gac?tac?tac?atg?agc?TGG?ATC?CGC?CAG?GCT?CCA?GGG?AAG?taa?ataaaa?tat?aag?aca?ggc
3’-5’(SEQ?ID?NO：9)gcc?tgt?ctt?ata?ttt?tat?tta?CTT?CCC?TGG?AGC?CTG?GCG?GAT?CCA?gct?cat?gta?gtagtc?act?gaa?ggt
For the third time:
5’-3’(SEQ?ID?NO：10)tgg?atc?cgc?cag?gct?cca?ggg?aag?GGG?CTG?GAG?TGG?GTT?TCA?GAT?GAG?taaata?aaa?tat?aag?aca?ggc
3’-5’(SEQ?ID?NO：11)gcc?tgt?ctt?ata?ttt?tat?tta?CTC?ATC?TGA?AAC?CCA?CTC?CAG?CCC?ctt?ccc?tggagc?ctg?gcg?gat?cca
The 4th time
5’-3’(SEQ?ID?NO：12)ggg?ctg?gag?tgg?gtt?tca?gat?gag?GCT?GAC?TAT?TAC?TGT?AAC?TCC?CGGGAC?AGC?taa?ata?aaa?tat?aag?aca?ggc
3’-5’(SEQ?ID?NO：13)gcc?tgt?ctt?ata?ttt?tat?tta?GCT?GTC?CCG?GGA?GTT?ACA?GTA?ATA?GTC?AGCctc?atc?tga?aac?cca?ctc?cag?ccc
[embodiment 2]
The gene of G28-V50 and E216-S228 peptide chain among the anti-HBsAg scFv of the GenBank AF236816 login method according to embodiment 1 is connected on the carboxyl terminal in colicin Ia channel architecture territory (K544-I622), be prepared into the plasmid of the anti-HBV engineered polypeptide of artificial combination, the plasmid transfection that sudden change is good is in the engineering bacteria that does not contain plasmid, bacterium is bred in a large number (4L FB train the base, 225rpm, 37 ℃; 6h), (4 ℃ of centrifugation thalline, 6000g, 20min), get 4 ℃, 50mM borate buffer+2mM EDTA+2mM DTT 50-80ml suspension thalline, and ultrasonication (4 ℃, 40W, 1-2min), the broken thalline of high speed centrifugation (4 ℃, 70000g, 1.5h), get supernatant and add Vetstrep deposit D NA, 4 ℃, after 50mM borate buffer+2mM EDTA+2mM DTT 2L dialysed overnight, be splined on the CM ion exchange column, just obtained the purifying rate behind the 0.1-0.3M NaCl+50mM borate buffer gradient elution and reached anti-HBV engineered polypeptide more than 90%, about molecular weight 1.5 ten thousand, the crude preparation by using protein content is about 1.5mg/ml.In the preliminary experiment, this project polypeptide does not show toxic side effect to the human body cell and the laboratory animal of vitro culture.
[embodiment 3]
According to the method for embodiment 1, the aminoterminal with the anti-HBV PreSl scFv of GenBank AF427148 login is connected to the water-soluble pore passage structure of colicin Ia territory forms recombinant plasmid, the recombinant polypeptide of acquisition molecular weight about 40,000;
The anti-HBV PreSl scFv of GenBank AF427148 login is connected to the carboxyl terminal in the water-soluble pore passage structure of colicin Ia territory, obtains the recombinant polypeptide of molecular weight about 40,000;
The anti-HBV PreSl scFv of GenBank AF427148 login is connected to the aminoterminal of colicin Ia, obtains the recombinant polypeptide of molecular weight about 90,000;
The anti-HBV PreSl scFv of GenBank AF427148 login is connected to the carboxyl terminal of colicin Ia, obtains the recombinant polypeptide of molecular weight about 90,000.
[embodiment 4] reorganization antiviral polypeptide is to the lethal effect of the HBV of vitro culture
With the human liver cell strain HepG2 2.2.15 of HBV transfected gene (Dr.C Wu (UCHC, Connecticut USA) provide) cell culture fluid is through 100,000g, 3hr handles the complete HBV virus that this culturing cell of enrichment produces (Dane ' s particle) for 4 ℃.The nutrient solution that will contain a large amount of HBV is with the anti-HBV engineered polypeptide incubated at room of 5: 1 capacity ratios and embodiment 1 preparation after 1 hour, peek microlitre sample places on the copper mesh and amplify 50,000 times of observations with transmission electron microscope after the dyeing of 1% phospho-wolframic acid, see that the interior dyeing of HBV after the processing deepens, and confirms that the HBV coating is destroyed by this polypeptide.Experimental result is asked for an interview Fig. 2.
Anti-HBV effect in the animal body of [embodiment 5] reorganization antiviral polypeptide
The structure of animal model (Dr.G Wu, UCHC, Connecticut USA provides, reference: Wu, C et.al, Hepatitis B virus infection of transplanted Human hepacytes causes abiochemical and histological hepatitis in immunocompetent rate W.J.ofGastroenterology, 9 (5): 978-983,2003, May 9):
1, tire mouse abdominal injection human liver cell
With the female mouse anesthesia of pregnant 15-17 days SD, open abdomen and expose gravid uterus, pass Uterus wall under the high light transmission and contain 1 * 10 in the injection of tire mouse intraperitoneal
5The PBS10 microlitre of human liver cell (HepG22.2.15).
2, human liver cell is transplanted
Neonate rat took place in 24 hours, and spleen injects and contains 2 * 10
6PBS 200 microlitres of human liver cell (the strain name is the same)
3, inject HBV
After one week of Transplanted cells, transplant and inject 105HBV particulate TEN (0.02MTris-Hcl in the successful mouse spleen, PH7.5,1mM EDTA, 0.15M Nacl) 100 microlitres, peripheral blood detects HBsAg, HBV DNA, hepatic tissue detects HbcAg, HBV RNA, and HBV cccDNA and histology change with conclusive evidence people-mouse liver inosculating model makes successfully.
Choose 9 on the chimeric in vivo of the mouse people-liver cell model that biochemical/histology hepatitis performance occurred, be divided into 3 groups, first group is control group a (n=3), and the abdominal cavity injects 0.9% physiological saline 1ml, 1 time weekly; Second group is control group b (n=3), and the abdominal cavity injects wild-type colicin Ia 0.5mg, 1 time weekly; The 3rd group is experimental group (n=3), and the abdominal cavity injects the anti-HBV polypeptide 0.5mg of embodiment 1 preparation, 1 time weekly.The 1st week, the 4th week and detect serum HBsAg (EIA method) and anti-HBc antibody (ELISA method, reorganization HbcAg bag is by 96 orifice plates) the 8th week respectively, the results are shown in Figure 3 and table 1 to estimate the drug effect of the anti-HBV polypeptide for preparing.As can be seen from Figure 4, to rising in the 4th week, mice serum HBsAg and the anti-HBc antibody of having injected anti-HBV polypeptide all significantly reduce.
The terminal point titre of table 1 is respectively organized serum anti--HBc
| Group | Sampling time | ||
| 0 week | 2 weeks | 4 weeks | |
| The polypeptide experimental group | ?36450 | ?12150 | ?4050 |
| Control group a | ?36450 | ?109350 | ?109350 |
| Control group b | ?36450 | ?109350 | ?109350 |
By above experimental result as seen, reorganization antiviral polypeptide of the present invention all shows higher anti-hepatitis B virus activities in external and animal body.
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
SEQUENCE LISTING＜110〉＜120〉＜130〉03P400065＜160〉13＜170〉PatentIn version 3.1＜210〉1＜211〉1878＜212〉DNA＜213〉Escherichia coli＜220〉＜221〉misc feature＜222〉 ( 1 ) .. ( 1878 )＜223〉Ia＜400〉1atgtctgacc ctgtacgtat tacaaatccc ggtgcagaat cgctggggta tgattcagat 60ggccatgaaa ttatggccgt tgatatttat gtaaaccctc cacgtgtcga tgtctttcat 120ggtaccccgc ctgcatggag ttccttcggg aacaaaacca tctggggcgg aaacgagtgg 180gttgatgatt ccccaacccg aagtgatatc gaaaaaaggg acaaggaaat cacagcgtac 240aaaaacacgc tcagcgcgca gcagaaagag aatgagaata agcgtactga agccggaaaa 300cgcctctctg cggcgattgc tgcaagggaa aaagatgaaa acacactgaa aacactccgt 360gccggaaacg cagatgccgc tgatattaca cgacaggagt tcagactcct gcaggcagag 420ctgagagaat acggattccg tactgaaatc gccggatatg acgccctccg gctgcataca 480gagagccgga tgctgtttgc tgatgctgat tctcttcgta tatctccccg ggaggccagg 540tcgttaatcg aacaggctga aaaacggcag aaggatgcgc agaacgcaga caagaaggcc 600gctgatatgc ttgctgaata cgagcgcaga aaaggtattc tggacacccg gttgtcagag 660ctggaaaaaa atggcggggc agcccttgcc gttcttgatg cacaacaggc ccgtctgctc 720gggcagcaga cacggaatga cagggccatt tcagaggccc ggaataaact cagttcagtg 780acggaatcgc ttaacacggc ccgtaatgca ttaaccagag ctgaacaaca gctgacgcaa 840cagaaaaaca cgcctgacgg caaaacgata gtttcccctg aaaaattccc ggggcgttca 900tcaacaaatc attctattgt tgtgagcggt gatccgagat ttgccggtac gataaaaatc 960acaaccagcg cagtcatcga taaccgtgca aacctgaatt atcttctgag ccattccggt 1020ctggactata aacgcaatat tctgaatgac cggaatccgg tggtgacaga ggatgtggaa 1080ggtgacaaga aaatttataa tgctgaagtt gctgaatggg ataagttacg gcaaagattg 1140cttgatgcca gaaataaaat cacctctgct gaatctgcgg taaattcggc gagaaataac 1200ctcagtgcca gaacaaatga gcaaaagcat gcaaatgacg ctcttaatgc cctgttgaag 1260gaaaaagaga atatacgtaa ccagctttcc ggcatcaatc agaagatagc ggaagagaaa 1320agaaaacagg atgaactgaa ggcaacgaaa gacgcaatta atttcacaac agagttcctg 1380aaatcagttt cagaaaaata tggtgcaaaa gctgagcagt tagccagaga gatggccggg 1440caggctaaag ggaagaaaat acgtaatgtt gaagaggcat taaaaacgta tgaaaagtac 1500cgggctgaca ttaacaaaaa aattaatgca aaagatcgtg cagcgattgc cgcagccctt 1560gagtctgtga agctgtctga tatatcgtct aatctgaaca gattcagtcg gggactggga 1620tatgcaggaa aatttacaag tcttgctgac tggatcactg agtttggtaa ggctgtccgg 1680acagagaact ggcgtcctct ttttgttaaa acagaaacca tcatagcagg caatgccgca 1740acggctcttg tggcactggt cttcagtatt cttaccggaa gcgctttagg cattatcggg 1800tatggtttac tgatggctgt caccggtgcg ctgattgatg aatcgcttgt ggaaaaagcg 1860aataagttct ggggtatt 1878＜210〉2＜211〉108＜212〉DNA＜213〉＜220〉＜221〉CDS＜222〉 ( 1 ) .. ( 108 )＜223〉HBsAgG28-V50E216-S228＜400〉2gga ttc acc ttc agt gac tac tac atg agc tgg atc cgc cag gct cca 48Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro1 5 10 15ggg aag ggg ctg gag tgg gtt tca gat gag gct gac tat tac tgt aac 96Gly Lys Gly Leu Glu Trp Val Ser Asp Glu Ala Asp Tyr Tyr Cys Asn
20??????????????????25??????????????????30tcc?cgg?gac?agc????????????????????????????????????????????????????108Ser?Arg?Asp?Ser
35＜210〉3＜211〉36＜212〉PRT＜213〉artificial sequence＜400〉3Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro1,5 10 15Gly Lys Gly Leu Glu Trp Val Ser Asp Glu Ala Asp Tyr Tyr Cys Asn
20??????????????????25??????????????????30Ser?Arg?Asp?Ser
35＜210〉4＜211〉1986＜212〉DNA＜213〉artificial sequence＜220〉＜221〉CDS＜222〉(1) .. (1986)＜223〉coding anti-hepatitis virus polypeptide＜400〉4atg tct gac cct gta cgt att aca aat ccc ggt gca gaa tcg ctg ggg 48Met Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1,5 10 15tat gat tca gat ggc cat gaa att atg gcc gtt gat att tat gta aac 96Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn
20??????????????????25??????????????????30cct?cca?cgt?gtc?gat?gtc?ttt?cat?ggt?acc?ccg?cct?gca?tgg?agt?tcc??????144Pro?Pro?Arg?Val?Asp?Val?Phe?His?Gly?Thr?Pro?Pro?Ala?Trp?Ser?Ser
35??????????????????40??????????????????45ttc?ggg?aac?aaa?acc?atc?tgg?ggc?gga?aac?gag?tgg?gtt?gat?gat?tcc??????192Phe?Gly?Asn?Lys?Thr?Ile?Trp?Gly?Gly?Asn?Glu?Trp?Val?Asp?Asp?Ser
50??????????????????55??????????????????60cca?acc?cga?agt?gat?atc?gaa?aaa?agg?gac?aag?gaa?atc?aca?gcg?tac??????240Pro?Thr?Arg?Ser?Asp?Ile?Glu?Lys?Arg?Asp?Lys?Glu?Ile?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80aaa?aac?acg?ctc?agc?gcg?cag?cag?aaa?gag?aat?gag?aat?aag?cgt?act??????288Lys?Asn?Thr?Leu?Ser?Ala?Gln?Gln?Lys?Glu?Asn?Glu?Asn?Lys?Arg?Thr
85??????????????????90??????????????????95gaa?gcc?gga?aaa?cgc?ctc?tct?gcg?gcg?att?gct?gca?agg?gaa?aaa?gat??????336Glu?Ala?Gly?Lys?Arg?Leu?Ser?Ala?Ala?Ile?Ala?Ala?Arg?Glu?Lys?Asp
100?????????????????105?????????????????110gaa?aac?aca?ctg?aaa?aca?ctc?cgt?gcc?gga?aac?gca?gat?gcc?gct?gat??????384Glu?Asn?Thr?Leu?Lys?Thr?Leu?Arg?Ala?Gly?Asn?Ala?Asp?Ala?Ala?Asp
115?????????????????120?????????????????125att?aca?cga?cag?gag?ttc?aga?ctc?ctg?cag?gca?gag?ctg?aga?gaa?tac??????432Ile?Thr?Arg?Gln?Glu?Phe?Arg?Leu?Leu?Gln?Ala?Glu?Leu?Arg?Glu?Tyr
130?????????????????135?????????????????140gga?ttc?cgt?act?gaa?atc?gcc?gga?tat?gac?gcc?ctc?cgg?ctg?cat?aca??????480Gly?Phe?Arg?Thr?Glu?Ile?Ala?Gly?Tyr?Asp?Ala?Leu?Arg?Leu?His?Thr145?????????????????150?????????????????155?????????????????160gag?agc?cgg?atg?ctg?ttt?gct?gat?gct?gat?tct?ctt?cgt?ata?tct?ccc??????528Glu?Ser?Arg?Met?Leu?Phe?Ala?Asp?Ala?Asp?Ser?Leu?Arg?Ile?Ser?Pro
165?????????????????170?????????????????175cgg?gag?gcc?agg?tcg?tta?atc?gaa?cag?gct?gaa?aaa?cgg?cag?aag?gat??????576Arg?Glu?Ala?Arg?Ser?Leu?Ile?Glu?Gln?Ala?Glu?Lys?Arg?Gln?Lys?Asp
180?????????????????185?????????????????190gcg?cag?aac?gca?gac?aag?aag?gcc?gct?gat?atg?ctt?gct?gaa?tac?gag??????624Ala?Gln?Asn?Ala?Asp?Lys?Lys?Ala?Ala?Asp?Met?Leu?Ala?Glu?Tyr?Glu
195?????????????????200?????????????????205cgc?aga?aaa?ggt?att?ctg?gac?acc?cgg?ttg?tca?gag?ctg?gaa?aaa?aat??????672Arg?Arg?Lys?Gly?Ile?Leu?Asp?Thr?Arg?Leu?Ser?Glu?Leu?Glu?Lys?Asn
210?????????????????215?????????????????220ggc?ggg?gca?gcc?ctt?gcc?gtt?ctt?gat?gca?caa?cag?gcc?cgt?ctg?ctc??????720Gly?Gly?Ala?Ala?Leu?Ala?Val?Leu?Asp?Ala?Gln?Gln?Ala?Arg?Leu?Leu225?????????????????230?????????????????235?????????????????240ggg?cag?cag?aca?cgg?aat?gac?agg?gcc?att?tca?gag?gcc?cgg?aat?aaa??????768Gly?Gln?Gln?Thr?Arg?Asn?Asp?Arg?Ala?Ile?Ser?Glu?Ala?Arg?Asn?Lys
245?????????????????250?????????????????255ctc?agt?tca?gtg?acg?gaa?tcg?ctt?aac?acg?gcc?cgt?aat?gca?tta?acc??????816Leu?Ser?Ser?Val?Thr?Glu?Ser?Leu?Asn?Thr?Ala?Arg?Asn?Ala?Leu?Thr
260?????????????????265?????????????????270aga?gct?gaa?caa?cag?ctg?acg?caa?cag?aaa?aac?acg?cct?gac?ggc?aaa??????864Arg?Ala?Glu?Gln?Gln?Leu?Thr?Gln?Gln?Lys?Asn?Thr?Pro?Asp?Gly?Lys
275?????????????????280?????????????????285acg?ata?gtt?tcc?cct?gaa?aaa?ttc?ccg?ggg?cgt?tca?tca?aca?aat?cat??????912Thr?Ile?Val?Ser?Pro?Glu?Lys?Phe?Pro?Gly?Arg?Ser?Ser?Thr?Asn?His
290?????????????????295?????????????????300tct?att?gtt?gtg?agc?ggt?gat?ccg?aga?ttt?gcc?ggt?acg?ata?aaa?atc??????960Ser?Ile?Val?Val?Ser?Gly?Asp?Pro?Arg?Phe?Ala?Gly?Thr?Ile?Lys?Ile305?????????????????310?????????????????315?????????????????320aca?acc?agc?gca?gtc?atc?gat?aac?cgt?gca?aac?ctg?aat?tat?ctt?ctg?????1008Thr?Thr?Ser?Ala?Val?Ile?Asp?Asn?Arg?Ala?Asn?Leu?Asn?Tyr?Leu?Leu
325?????????????????330?????????????????335agc?cat?tcc?ggt?ctg?gac?tat?aaa?cgc?aat?att?ctg?aat?gac?cgg?aat?????1056Ser?His?Ser?Gly?Leu?Asp?Tyr?Lys?Arg?Asn?Ile?Leu?Asn?Asp?Arg?Asn
340?????????????????345?????????????????350ccg?gtg?gtg?aca?gag?gat?gtg?gaa?ggt?gac?aag?aaa?att?tat?aat?gct?????1104Pro?Val?Val?Thr?Glu?Asp?Val?Glu?Gly?Asp?Lys?Lys?Ile?Tyr?Asn?Ala
355?????????????????360?????????????????365gaa?gtt?gct?gaa?tgg?gat?aag?tta?cgg?caa?aga?ttg?ctt?gat?gcc?aga?????1152Glu?Val?Ala?Glu?Trp?Asp?Lys?Leu?Arg?Gln?Arg?Leu?Leu?Asp?Ala?Arg
370?????????????????375?????????????????380aat?aaa?atc?acc?tct?gct?gaa?tct?gcg?gta?aat?tcg?gcg?aga?aat?aac?????1200Asn?Lys?Ile?Thr?Ser?Ala?Glu?Ser?Ala?Val?Asn?Ser?Ala?Arg?Asn?Asn385?????????????????390?????????????????395?????????????????400ctc?agt?gcc?aga?aca?aat?gag?caa?aag?cat?gca?aat?gac?gct?ctt?aat?????1248Leu?Ser?Ala?Arg?Thr?Asn?Glu?Gln?Lys?His?Ala?Asn?Asp?Ala?Leu?Asn
405?????????????????410?????????????????415gcc?ctg?ttg?aag?gaa?aaa?gag?aat?ata?cgt?aac?cag?ctt?tcc?ggc?atc?????1296Ala?Leu?Leu?Lys?Glu?Lys?Glu?Asn?Ile?Arg?Asn?Gln?Leu?Ser?Gly?Ile
420?????????????????425?????????????????430aat?cag?aag?ata?gcg?gaa?gag?aaa?aga?aaa?cag?gat?gaa?ctg?aag?gca?????1344Asn?Gln?Lys?Ile?Ala?Glu?Glu?Lys?Arg?Lys?Gln?Asp?Glu?Leu?Lys?Ala
435?????????????????440?????????????????445acg?aaa?gac?gca?att?aat?ttc?aca?aca?gag?ttc?ctg?aaa?tca?gtt?tca?????1392Thr?Lys?Asp?Ala?Ile?Asn?Phe?Thr?Thr?Glu?Phe?Leu?Lys?Ser?Val?Ser
450?????????????????455?????????????????460gaa?aaa?tat?ggt?gca?aaa?gct?gag?cag?tta?gcc?aga?gag?atg?gcc?ggg?????1440Glu?Lys?Tyr?Gly?Ala?Lys?Ala?Glu?Gln?Leu?Ala?Arg?Glu?Met?Ala?Gly465?????????????????470?????????????????475?????????????????480cag?gct?aaa?ggg?aag?aaa?ata?cgt?aat?gtt?gaa?gag?gca?tta?aaa?acg?????1488Gln?Ala?Lys?Gly?Lys?Lys?Ile?Arg?Asn?Val?Glu?Glu?Ala?Leu?Lys?Thr
485?????????????????490?????????????????495tat?gaa?aag?tac?cgg?gct?gac?att?aac?aaa?aaa?att?aat?gca?aaa?gat?????1536Tyr?Glu?Lys?Tyr?Arg?Ala?Asp?Ile?Asn?Lys?Lys?Ile?Asn?Ala?Lys?Asp
500?????????????????505?????????????????510cgt?gca?gcg?att?gcc?gca?gcc?ctt?gag?tct?gtg?aag?ctg?tct?gat?ata?????1584Arg?Ala?Ala?Ile?Ala?Ala?Ala?Leu?Glu?Ser?Val?Lys?Leu?Ser?Asp?Ile
515?????????????????520?????????????????525tcg?tct?aat?ctg?aac?aga?ttc?agt?cgg?gga?ctg?gga?tat?gca?gga?aaa?????1632Ser?Ser?Asn?Leu?Asn?Arg?Phe?Ser?Arg?Gly?Leu?Gly?Tyr?Ala?Gly?Lys
530?????????????????535?????????????????540ttt?aca?agt?ctt?gct?gac?tgg?atc?act?gag?ttt?ggt?aag?gct?gtc?cgg?????1680Phe?Thr?Ser?Leu?Ala?Asp?Trp?Ile?Thr?Glu?Phe?Gly?Lys?Ala?Val?Arg545?????????????????550?????????????????555?????????????????560aca?gag?aac?tgg?cgt?cct?ctt?ttt?gtt?aaa?aca?gaa?acc?atc?ata?gca?????1728Thr?Glu?Asn?Trp?Arg?Pro?Leu?Phe?Val?Lys?Thr?Glu?Thr?Ile?Ile?Ala
565?????????????????570?????????????????575ggc?aat?gcc?gca?acg?gct?ctt?gtg?gca?ctg?gtc?ttc?agt?att?ctt?acc?????1776Gly?Asn?Ala?Ala?Thr?Ala?Leu?Val?Ala?Leu?ValPhe?Ser?Ile?Leu?Thr
580?????????????????585????????????????590gga?agc?gct?tta?ggc?att?atc?ggg?tat?ggt?tta?ctg?atg?gct?gtc?acc?????1824Gly?Ser?Ala?Leu?Gly?Ile?Ile?Gly?Tyr?Gly?Leu?Leu?Met?Ala?Val?Thr
595?????????????????600?????????????????605ggt?gcg?ctg?att?gat?gaa?tcg?ctt?gtg?gaa?aaa?gcg?aat?aag?ttc?tgg????1872Gly?Ala?Leu?Ile?Asp?Glu?Ser?Leu?Val?Glu?Lys?Ala?Asn?Lys?Phe?Trp
610?????????????????615?????????????????620ggt?att?gga?ttc?acc?ttc?agt?gac?tac?tac?atg?agc?tgg?atc?cgc?cag????1920Gly?Ile?Gly?Phe?Thr?Phe?Ser?Asp?Tyr?Tyr?Met?Ser?Trp?Ile?Arg?Gln625?????????????????630?????????????????635?????????????????640gct?cca?ggg?aag?ggg?ctg?gag?tgg?gtt?tca?gat?gag?gct?gac?tat?tac????1968Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Asp?Glu?Ala?Asp?Tyr?Tyr
645?????????????????650?????????????????655tgt?aac?tcc?cgg?gac?agc????????????????????????????????????????????1986Cys?Asn?Ser?Arg?Asp?Ser
660＜210〉5＜211〉662＜212〉PRT＜213〉artificial sequence＜400〉5Met Ser Asp Pro Val Arg Ile Thr Asn Pro Gly Ala Glu Ser Leu Gly1,5 10 15Tyr Asp Ser Asp Gly His Glu Ile Met Ala Val Asp Ile Tyr Val Asn
20??????????????????25??????????????????30Pro?Pro?Arg?Val?Asp?Val?Phe?His?Gly?Thr?Pro?Pro?Ala?Trp?Ser?Ser
35??????????????????40??????????????????45Phe?Gly?Asn?Lys?Thr?Ile?Trp?Gly?Gly?Asn?Glu?Trp?Val?Asp?Asp?Ser
50??????????????????55??????????????????60Pro?Thr?Arg?Ser?Asp?Ile?Glu?Lys?Arg?Asp?Lys?Glu?Ile?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80Lys?Asn?Thr?Leu?Ser?Ala?Gln?Gln?Lys?Glu?Asn?Glu?Asn?Lys?Arg?Thr
85??????????????????90??????????????????95Glu?Ala?Gly?Lys?Arg?Leu?Ser?Ala?Ala?Ile?Ala?Ala?Arg?Glu?Lys?Asp
100?????????????????105?????????????????110Glu?Asn?Thr?Leu?Lys?Thr?Leu?Arg?Ala?Gly?Asn?Ala?Asp?Ala?Ala?Asp
115?????????????????120?????????????????125Ile?Thr?Arg?Gln?Glu?Phe?Arg?Leu?Leu?Gln?Ala?Glu?Leu?Arg?Glu?Tyr
130?????????????????135?????????????????140Gly?Phe?Arg?Thr?Glu?Ile?Ala?Gly?Tyr?Asp?Ala?Leu?Arg?Leu?His?Thr145?????????????????150?????????????????155?????????????????160Glu?Ser?Arg?Met?Leu?Phe?Ala?Asp?Ala?Asp?Ser?Leu?Arg?Ile?Ser?Pro
165?????????????????170?????????????????175Arg?Glu?Ala?Arg?Ser?Leu?Ile?Glu?Gln?Ala?Glu?Lys?Arg?Gln?Lys?Asp
180?????????????????185?????????????????190Ala?Gln?Asn?Ala?Asp?Lys?Lys?Ala?Ala?Asp?Met?Leu?Ala?Glu?Tyr?Glu
195?????????????????200?????????????????205Arg?Arg?Lys?Gly?Ile?Leu?Asp?Thr?Arg?Leu?Ser?Glu?Leu?Glu?Lys?Asn
210?????????????????215?????????????????220Gly?Gly?Ala?Ala?Leu?Ala?Val?Leu?Asp?Ala?Gln?Gln?Ala?Arg?Leu?Leu225?????????????????230?????????????????235?????????????????240Gly?Gln?Gln?Thr?Arg?Asn?Asp?Arg?Ala?Ile?Ser?Glu?Ala?Arg?Asn?Lys
245?????????????????250?????????????????255Leu?Ser?Ser?Val?Thr?Glu?Ser?Leu?Asn?Thr?Ala?Arg?Asn?Ala?Leu?Thr
260?????????????????265?????????????????270Arg?Ala?Glu?Gln?Gln?Leu?Thr?Gln?Gln?Lys?Asn?Thr?Pro?Asp?Gly?Lys
275?????????????????280?????????????????285Thr?Ile?Val?Ser?Pro?Glu?Lys?Phe?Pro?Gly?Arg?Ser?Ser?Thr?Asn?His
290?????????????????295?????????????????300Ser?Ile?Val?Val?Ser?Gly?Asp?Pro?Arg?Phe?Ala?Gly?Thr?Ile?Lys?Ile305?????????????????310?????????????????315?????????????????320Thr?Thr?Ser?Ala?Val?Ile?Asp?Asn?Arg?Ala?Asn?Leu?Asn?Tyr?Leu?Leu
325?????????????????330?????????????????335Ser?His?Ser?Gly?Leu?Asp?Tyr?Lys?Arg?Asn?Ile?Leu?Asn?Asp?Arg?Asn
340?????????????????345?????????????????350Pro?Val?Val?Thr?Glu?Asp?Val?Glu?Gly?Asp?Lys?Lys?Ile?Tyr?Asn?Ala
355?????????????????360?????????????????365Glu?Val?Ala?Glu?Trp?Asp?Lys?Leu?Arg?Gln?Arg?Leu?Leu?Asp?Ala?Arg
370?????????????????375?????????????????380Asn?Lys?Ile?Thr?Ser?Ala?Glu?Ser?Ala?Val?Asn?Ser?Ala?Arg?Asn?Asn385?????????????????390?????????????????395?????????????????400Leu?Ser?Ala?Arg?Thr?Asn?Glu?Gln?Lys?His?Ala?Asn?Asp?Ala?Leu?Asn
405?????????????????410?????????????????415Ala?Leu?Leu?Lys?Glu?Lys?Glu?Asn?Ile?Arg?Asn?Gln?Leu?Ser?Gly?Ile
420?????????????????425?????????????????430Asn?Gln?Lys?Ile?Ala?Glu?Glu?Lys?Arg?Lys?Gln?Asp?Glu?Leu?Lys?Ala
435?????????????????440?????????????????445Thr?Lys?Asp?Ala?Ile?Asn?Phe?Thr?Thr?Glu?Phe?Leu?Lys?Ser?Val?Ser
450?????????????????455?????????????????460Glu?Lys?Tyr?Gly?Ala?Lys?Ala?Glu?Gln?Leu?Ala?Arg?Glu?Met?Ala?Gly465?????????????????470?????????????????475?????????????????480Gln?Ala?Lys?Gly?Lys?Lys?Ile?Arg?Asn?Val?Glu?Glu?Ala?Leu?Lys?Thr
485?????????????????490?????????????????495Tyr?Glu?Lys?Tyr?Arg?Ala?Asp?Ile?Asn?Lys?Lys?Ile?Asn?Ala?Lys?Asp
500?????????????????505?????????????????510Arg?Ala?Ala?Ile?Ala?Ala?Ala?Leu?Glu?Ser?Val?Lys?Leu?Ser?Asp?Ile
515?????????????????520?????????????????525Ser?Ser?Asn?Leu?Asn?Arg?Phe?Ser?Arg?Gly?Leu?Gly?Tyr?Ala?Gly?Lys
530?????????????????535?????????????????540Phe?Thr?Ser?Leu?Ala?Asp?Trp?Ile?Thr?Glu?Phe?Gly?Lys?Ala?Val?Arg545?????????????????550?????????????????555?????????????????560Thr?Glu?Asn?Trp?Arg?Pro?Leu?Phe?Val?Lys?Thr?Glu?Thr?Ile?Ile?Ala
565?????????????????570?????????????????575Gly?Asn?Ala?Ala?Thr?Ala?Leu?Val?Ala?Leu?Val?Phe?Ser?Ile?Leu?Thr
580?????????????????585?????????????????590Gly?Ser?Ala?Leu?Gly?Ile?Ile?Gly?Tyr?Gly?Leu?Leu?Met?Ala?Val?Thr
595?????????????????600?????????????????605Gly?Ala?Leu?Ile?Asp?Glu?Ser?Leu?Val?Glu?Lys?Ala?Asn?Lys?Phe?Trp
610?????????????????615?????????????????620Gly?Ile?Gly?Phe?Thr?Phe?Ser?Asp?Tyr?Tyr?Met?Ser?Trp?Ile?Arg?Gln625?????????????????630?????????????????635?????????????????640Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ser?Asp?Glu?Ala?Asp?Tyr?Tyr
645?????????????????650?????????????????655Cys?Asn?Ser?Arg?Asp?Ser
660＜210〉6＜211〉72＜212〉DNA＜213〉＜220〉＜221〉misc feature＜222〉 ( 1 ) .. ( 72 )＜223〉＜400〉6gcgaataagt tctggggtat tggattcacc ttcagtgact actacatgag ctaaataaaa 60tataagacag gc 72＜210〉7＜211〉72＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 72 )＜223〉＜400〉7cgcttattca agaccccata acctaagtgg aagtcactga tgatgtactc gatttatttt 60atattctgtc cg 72＜210〉8＜211〉69＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 69 )＜223〉＜400〉8accttcagtg actactacat gagctggatc cgccaggctc cagggaagta aataaaatat 60aagacaggc 69＜210〉9＜211〉69＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 69 )＜223〉＜400〉9tggaagtcac tgatgatgta ctcgacctag gcggtccgag gtcccttcat ttattttata 60ttctgtccg 69＜210〉10＜211〉69＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 69 )＜223〉＜400〉10tggatccgcc aggctccagg gaaggggctg gagtgggttt cagatgagta aataaaatat 60aagacaggc 69＜210〉11＜211〉69＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 69 )＜223〉＜400〉11acctaggcgg tccgaggtcc cttccccgac ctcacccaaa gtctactcat ttattttata 60ttctgtccg 69＜210〉12＜211〉75＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 75 )＜223〉＜400〉12gggctggagt gggtttcaga tgaggctgac tattactgta actcccggga cagctaaata 60aaatataaga caggc 75＜210〉13＜211〉75＜212〉DNA＜213〉＜220〉＜221〉misc_feature＜222〉 ( 1 ) .. ( 75 )＜223〉＜400〉13cccgacctca cccaaagtct actccgactg ataatgacat tgagggccct gtcgatttat 60tttatattct gtccg 75
Claims (20)
1, the Nucleotide of coding reorganization antiviral polypeptide is characterized in that it contains
Coding can form the colicin of ionic channel or the gene in its water-based pore passage structure territory, and coding can with the antigen on the peplos or the gene of receptor-specific bonded polypeptide.
2, Nucleotide according to claim 1, the wherein said colicin that forms ionic channel is selected from colicin E1, Ia, Ib, A, B and N.
3, Nucleotide according to claim 1, wherein said virus are hepatitis B virus.
4, antigen or receptor-specific bonded polypeptide on the Nucleotide according to claim 3, wherein said and peplos are anti-HBV PreSl scFv, and its GenBank accession number is AF427148.
5, Nucleotide according to claim 4, wherein said anti-HBV PreSl scFv polypeptide chain is connected to the aminoterminal of described colicin Ia.
6, Nucleotide according to claim 4, wherein said anti-HBV PreSl scFv polypeptide chain is connected to the carboxyl terminal of described colicin Ia.
7, Nucleotide according to claim 4, wherein said anti-HBV PreSl scFv polypeptide chain is connected to the aminoterminal in described colicin Ia water-based pore passage structure territory.
8, Nucleotide according to claim 4, wherein said anti-HBV PreSl scFv polypeptide chain is connected to the carboxyl terminal in described colicin Ia water-based pore passage structure territory.
9, antigen or receptor-specific bonded polypeptide on the Nucleotide according to claim 1, wherein said and hepatitis B virus coating are HBsAg single-chain antibody scFv, and its GenBank accession number is AF236816.
10, one or several antigen binding domains that antigen on the Nucleotide according to claim 9, wherein said and hepatitis B virus coating or receptor-specific bonded polypeptide are HBsAg single-chain antibody scFv.
11, Nucleotide according to claim 10, the antigen binding domain of wherein said HBsAg single-chain antibody are G28-V50 and two sections peptide chains of E216-S228 of scFv.
12, Nucleotide according to claim 11, the aminoterminal of G28-V50 and two sections peptide chains of E216-S228 is connected in the carboxyl terminal of described colicin Ia in the wherein said anti-HBsAg single-chain antibody.
13, Nucleotide according to claim 11, the aminoterminal of G28-V50 and two sections peptide chains of E216-S228 is connected in the carboxyl terminal in described colicin Ia water-based pore passage structure territory in the wherein said anti-HBsAg single-chain antibody.
14, Nucleotide according to claim 12 wherein contains the nucleotide sequence of SEQ ID NO.4.
15, a kind of recombinant plasmid is characterized in that it contains
Coding can form the colicin of ionic channel or the gene in its water-based pore passage structure territory, and coding can with the antigen on the peplos or the gene of receptor-specific bonded polypeptide.
16, recombinant plasmid according to claim 15 wherein contains the nucleotide sequence of SEQ ID NO.4.
17, a kind of reorganization antiviral polypeptide, it contains colicin or its water-based pore passage structure territory that can form ionic channel, and can with antigen or the receptor-specific bonded polypeptide on the peplos.
18, antiviral polypeptide according to claim 17 wherein comprises the aminoacid sequence of SEQ ID NO.5.
19, the pharmaceutical composition that contains claim 17 or 18 described reorganization antiviral polypeptides.
20, the preparation method of reorganization antiviral polypeptide may further comprise the steps:
Coding can be operably connected with the gene that the gene of antigen on the peplos or receptor-specific bonded polypeptide and coding can form the colicin of ionic channel or its water-based pore passage structure territory obtain the recombinate gene of antiviral polypeptide of coding;
The gene that obtains imported in the expression system express;
The antiviral polypeptide that separates polypeptide expressed and obtain.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA031492142A CN1482242A (en) | 2002-06-17 | 2003-06-17 | A anti-enveloped virus recombinant polypeptide and its preparation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02113897A CN1396178A (en) | 2002-06-17 | 2002-06-17 | Engineered polypeptide resisting infection of coated virus (hepatitis B virus) and its preparing process |
| CN02113897.4 | 2002-06-17 | ||
| CNA031492142A CN1482242A (en) | 2002-06-17 | 2003-06-17 | A anti-enveloped virus recombinant polypeptide and its preparation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006103494A2 (en) * | 2004-12-10 | 2006-10-05 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| CN1314806C (en) * | 2005-01-14 | 2007-05-09 | 四川大学 | Miniaturized polypeptide of anti EB Virus tumour, application and preparation method |
| WO2007083175A1 (en) * | 2006-01-17 | 2007-07-26 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| WO2023050661A1 (en) * | 2021-09-28 | 2023-04-06 | 北京亦科信息菌素研究院有限公司 | Pheromonicin against sars-cov-2 and use thereof |
-
2003
- 2003-06-17 CN CNA031492142A patent/CN1482242A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006103494A2 (en) * | 2004-12-10 | 2006-10-05 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| WO2006103494A3 (en) * | 2004-12-10 | 2006-12-07 | West China Hospital Sichuan Univ | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| CN1314806C (en) * | 2005-01-14 | 2007-05-09 | 四川大学 | Miniaturized polypeptide of anti EB Virus tumour, application and preparation method |
| WO2007083175A1 (en) * | 2006-01-17 | 2007-07-26 | West China Hospital, Sichuan University | Antiviral bifunctional molecules, methods of construction and methods of treating virus-induced cancer therewith |
| WO2023050661A1 (en) * | 2021-09-28 | 2023-04-06 | 北京亦科信息菌素研究院有限公司 | Pheromonicin against sars-cov-2 and use thereof |
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