CN100523180C - Recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use - Google Patents
Recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase and its preparing method and use Download PDFInfo
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Abstract
The present invention provides the recombinant protein (HGXPRT) of malignant malarial parasite hypoxanthine-uanine-xanthine ribose phosphate transferase, the recombinant protein coding DNA sequence, vector containing the DNA sequence, host cell containing the vector, gene engineering process for preparing the recombinant protein, and the use of the recombinant protein. The recombinant protein HGXPRT has excellent immunogenicity and excellent enzymic kinetic characteristic, and can induce effective malarial parasite antagonizing immune response in the immunized individual and produce efficient enzymological activity.
Description
Technical field
The present invention relates to DNA recombinant technology and recombinant vaccine field.More specifically, the present invention relates to a kind of malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme (hypoxanthine-guanine-xanthine phosphoribosyltransferase that contains, HGXPRT) recombinant protein, the encode dna sequence dna of this recombinant protein, the carrier that contains this dna sequence dna, the host cell that contains this carrier prepares the method for this recombinant protein with genetically engineered, and this recombinant protein is in the application aspect drug target protein and development malaria vaccine.
Background technology
Malaria is one of the most ancient human transmissible disease, still has a strong impact on human beings'health at present.According to the latest survey of The World Health Organization (WHO), 40% of about world population still is among the malaria threat, is distributed in more than 100 country.There are every year 3 to 500,000,000 people to suffer from malaria at present in the world, wherein about 3,000,000 people's death.Even more serious is that because plasmodium and the drug-fast generation of mosquito matchmaker and diffusion rapidly, malaria not only is not effectively controlled, and the gesture of staging a comeback is arranged on the contrary.Therefore, carry out the plan of whole world containment malaria and become WHO at one of new millennium two priority research areass.
The three approach that mainly contains that people rely in malaria control or expect to obtain to break through: the anti-system of antimalarial drug, malaria vaccine and mosquito matchmaker.But the anti-system of antimalarial drug and mosquito matchmaker is faced with huge difficulty and challenge, and this is because plasmodium and drug-fast generation of mosquito and diffusion.Although there is not the malaria vaccine of using value to come out at present as yet, generally believe that the development malaria vaccine is human control and even the important channel of eliminating malaria.
Studies show that in a large number malaria can realize preventing by developing effective vaccine.Can protect afterwards challenge infection fully as volunteer with the immunity of deactivation sporozoite.Use the plasmodial recombinant antigen immune mouse of mouse for another example, also can protect plasmodial challenge infection of the same race afterwards fully.In addition, the malaria epidemiological study shows that also that die from malaria mainly is the crowd of no malaria immunizing power, as: children and non-malaria district crowd in the malaria district enter the malaria district, and seldom dead because of malaria the adult in malaria district.Inoculate effective vaccine as crowd, make it reach same immunizing power, so just can realize the target of prevention of malaria and reduction malaria mortality ratio with malaria district adult to these no immunizing power.Therefore, the malaria vaccine development has become the hot in nature some problem in the world today.
In malaria vaccine research, there is the research of two candidate vaccines once to be subjected to extensive concern, the one, anti-sporozoite vaccine.This vaccine can suppress sporozoite invasion liver cell.But poor effect in clinical trial afterwards.According to one's analysis, its major cause is that this vaccine only produces anti-sporozoite immunizing power, attacks and survives even there are extremely indivedual sporozoites to escape this vaccine immunity like this, just can invade and breed at liver cell growth, produces enough merozoites and causes that the host is pathogenic.Another is a SPf.66 multivalence synthetic peptide vaccine.This vaccine is one and has only 3 10 peptide epitopes by the hydrophobic amino acid polymer [Patarroyo of 45 peptides of being formed by connecting of being separated by, M.E., et al, induction of protective immunity against experimental infection with malaria suingSynthetic peptides, Nature, 328:629,1987].This vaccine had once been obtained immune protective effect preferably in the douroucouli challenge trial, but failed to obtain the ideal clinical effectiveness afterwards in the test in place of Africa, South East Asia and South America, did not have using value.The reason of this vaccine failure may be that SPf.66 has only 45 peptides, and in short peptide sequence like this, may not have enough t cell epitopes can combine with the individual MHC molecule of majority, causes angtigen presentation to be obstructed.
Though plasmodial whole machine body can be finished the repertoire of vital movement only by a cellularity.Yet occurring in nature comprise plasmodium nearly 65000 surplus kind of protozoon all have unique metabolic characteristics, promptly all can not be by synthetic self the purine nucleotides of the approach of de novo synthesis purine ribonucleotide.In order to survive, plasmodium must pass through the purine salvage pathway, utilizes the purine bases and the Nucleotide purine biosynthesis Nucleotide of the interior moulding of purine salvage enzymes catalysis host cell of self.Known at present, (hypoxanine-guanine-xanine-phosphoribosyltransferaseHGXPRT) is elementary enzyme and a key enzyme of remedying purinase in the plasmodium body to hypoxanthine-guanine-xanthine phosphoribosyl transferase.Yet, for certain unknown cause, the report of the present HGXPRT that does not still successfully express.Therefore, people think that always HGXPRT can't be as antimalarial effective immunogen.
In sum, although carried out nearly 20 years, still do not have the vaccine that using value is arranged so far and come out based on the malaria vaccine research of biotechnology.
Therefore, this area presses for exploitation antimalarial effective immunogen and relevant vaccine.
Summary of the invention
One object of the present invention just provides a kind of new immunogen that can be used for malaria vaccine.Described immunogen is the recombinant protein that contains malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme (HGXPRT).
An object of the present invention is to provide this immunogenic method for making, purposes and contain this immunogenic vaccine composition.
In a first aspect of the present invention, a kind of recombinant protein is provided, it comprises the aminoacid sequence of malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme HGXPRT, and has the ribosyl forwarding function.More preferably, described recombinant protein has the aminoacid sequence shown in 1-231 position among the SEQ ID NO:2 or the 1-237 position.
In a second aspect of the present invention, provide a kind of isolated DNA molecule, its above-mentioned recombinant protein of the present invention of encoding.More preferably, described dna molecular is selected from down group: (a) have the nucleotide sequence shown in the 25-735 position among the SEQ ID NO:1; (b) has the nucleotide sequence shown in the 1-757 position among the SEQ ID NO:1.
In third aspect present invention, the carrier that contains above-mentioned dna molecular is provided, and the host cell that contains described carrier.
In a fourth aspect of the present invention, a kind of method that produces recombinant protein of the present invention is provided, it comprises step:
Under the condition that is fit to the described recombinant protein of expression, cultivate above-mentioned host cell, thereby give expression to described recombinant protein; With separate described recombinant protein.
In a fifth aspect of the present invention, a kind of composition is provided, it contains recombinant protein of the present invention or its coding DNA molecule and pharmaceutically acceptable carrier.
In another preference, described composition is a vaccine composition.
In a sixth aspect of the present invention, a kind of antibody is provided, it is incorporated into recombinant protein of the present invention specifically.
In a seventh aspect of the present invention, the purposes of a kind of recombinant protein of the present invention or malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme (HGXPRT) is provided, it is used to prepare the prevention of malaria vaccine.
Description of drawings
Fig. 1 is the synthesis strategy synoptic diagram of recombinant protein gene Pfhgxprt.
Synthesizing of Pfhgxprt gene: the 757bpPfhgxprt gene is divided into 8 oligonucleotide fragments, with asymmetric PCR method proceed step by step gene splicing.Gene 5 ' and 3 ' adds that respectively XhoI and EcoRI site are used for the clone of gene fragment.Synthetic fragment is cloned in the pBluscript carrier and carries out sequential analysis.
Fig. 2 is the agarose gel electrophoresis figure of total length Pfhgxprt gene.
After cutting with XhoI and EcoRI enzyme, electrophoresis showed Pfhgxprt synthetic gene band (swimming lane 1).
Fig. 3 is the expression that SDS-PAGE measures PfHGXPRT.
Inducing preceding Ohr (swimming lane 2) and inducing back 24 (swimming lanes 3), 48 (swimming lanes 4) and 72 hours (swimming lane 5) to get culture supernatant 10 μ l and carry out that SDS-PAGE separates and coomassie brilliant blue staining.The albumen that occurs clauses and subclauses at the 26KD place.
Fig. 4 is the SDS-PAGE electrophorogram of the PfHGXPRT recombinant protein of purifying.
After expressing the supernatant separation,, obtain target protein purity greater than 90% through SPS-PAGE mensuration with Ni-NTA post, molecular sieve, three step of cation seperation column purifying.Recombinant protein detects protein concentration through the Lowry method.Swimming lane 1:14 μ g; Swimming lane 2:28 μ g; Swimming lane 3:42 μ g; Swimming lane 4:56 μ g; Swimming lane 5:80 μ g;
Fig. 5 has shown the level of measuring the special IgG of immune mouse serum PfHGXPRT with the ELISA method.
Be ISA720 adjuvant+PfHGXPRT group, adjuvant and blank group antibody titers all＜1:50.
Fig. 6 has shown the level of measuring the special IgG of immune mouse serum PfHGXPRT with the IFA method.
Two groups are respectively: PfHGXPRT immunization experiment group mice serum (A); Negative control group mice serum (B)
The variation of parasitemia densities behind Fig. 7 PfHGXPRT immune mouse challenge infection
Behind the challenge infection 5 days, promptly mouse occurred before the death, infected the ratio of number and inspection mouse number again by the red corpuscle of daily inspection, obtained its average infection rate.Make figure with average infection rate.Wherein the I group is ISA720+PfHGXPRT; The II group is freund adjuvant+PfHGXPRT; The III group is ISA720 adjuvant control group; The IV group is the freund adjuvant control group; The V group is the blank group.
Embodiment
The inventor is through deeply and extensive studies, by to the transformation of HGXPRT codon and to the optimization of expression system, successfully made the recombinant protein of hypoxanthine-guanine-xanthine phosphoribosyl transferase HGXPRT first in Yeast system.Test shows, this recombinant protein can cause immunne response effectively, has the enzymic activity that ribosyl shifts, and is similar to the native conformation of HGXPRT on conformation.Finished the present invention on this basis.
HGXPRT is a kind of purine salvage enzymes, plays an important role in the anabolic salvage pathway of plasmodium purine.The molecular weight of HGXPRT is 26Kda.
As used herein, term " hypoxanthine-guanine-xanthine phosphoribosyl transferase ", " hypoxanthine-guanine-xanthine phosphoribosyl transferase HGXPRT " or " HGXPRT ", " PfHGXPRT " are used interchangeably, and all refer to hypoxanthine-guanine-xanthine phosphoribosyl transferase.The sequence of HGXPRT is known, for example GenBank accession number P20035.In addition, hypoxanthine-guanine-xanthine phosphoribosyl transferase is also sometimes referred to as " xanthoglobulin-guanine phosphoribosyltransferase ".
As used herein, term " recombinant protein of hypoxanthine-guanine-xanthine phosphoribosyl transferase ", " HGXPRT recombinant protein " etc. are used interchangeably, and all are meant the albumen that the aminoacid sequence by malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme HGXPRT constitutes.In addition, the recombinant protein of hypoxanthine-guanine-xanthine phosphoribosyl transferase comprises and contains or do not contain signal peptide, and the recombinant protein that contains or do not contain initial methionine.
As used herein, " aminoacid sequence of plasmodium hypoxanthine-guanine-xanthine phosphoribosyl transferase HGXPRT " refers to the aminoacid sequence in recombinant protein of the present invention in the term recombinant protein, this sequence has substantially the same aminoacid sequence with plasmodium HGXPRT natural or variation, and has biologic activity such as antigenic activity substantially the same with natural plasmodium HGXPRT and ribosyl forwarding function.Preferred plasmodium HGXPRT aminoacid sequence comprises (but being not limited to): the aminoacid sequence of natural PfHGXPRT, and eliminated this aminoacid sequence of glycosylation site.
In addition, also can add the aminoacid sequence that other do not influence biological characteristicses such as HGXPRT immunogenicity and red corpuscle combined function at the N of HGXPRT recombinant protein end or C-terminal.Preferably, the aminoacid sequence of these interpolations helps expressing (as signal peptide), help purifying (as 6xHis sequence, yeast saccharomyces cerevisiae α-factor signal peptide cleavage site (Glu-Lys-Arg)), maybe can promote the immunogenicity (for example sequence of cytokine such as IL-2, GM-CSF) of HGXPRT recombinant protein.
The dna sequence dna of code book invention recombinant protein, all synthetic.Also available pcr amplification or synthetic method obtain the DNA sequences encoding of HGXPRT, form the dna sequence dna of code book invention recombinant protein.
In order to improve the expression amount of host cell, can transform HGXPRT recombinant protein encoding sequence, for example adopt the codon of host cell preference, eliminate the sequence that is unfavorable for genetic transcription and translation.In one embodiment of the invention, just adopt the codon of yeast preference, HGXPRT recombinant protein gene is detected, eliminating is unfavorable for the sequence of genetic transcription and translation in gene, comprise the intron shearing site, transcription termination sequence etc., thus obtained to be beneficial to the encoding sequence of in yeast, expressing.
After the dna sequence dna that has obtained code book invention recombinant protein, it is connected into suitable expression vector, change proper host cell again over to.At last, cultivate the host cell after transforming, obtain new recombinant protein of the present invention by separation and purification.
As used herein, term " carrier " comprises plasmid, clay, expression vector, cloning vector, virus vector etc.
In the present invention, can select various carrier known in the art such as commercially available carrier for use.Such as, select commercially available carrier for use, the nucleotide sequence of then code book being invented new recombinant protein operationally is connected in expression regulation sequence, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, more preferably is yeast cell.
After obtaining transformed host cells, can under the condition that is fit to expression recombinant protein of the present invention, cultivate this cell, thereby give expression to recombinant protein.And then isolate the recombinant protein of expression.
On the other hand, the present invention comprises that also HGXPRT recombinant protein or its coding DNA are had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into HGXPRT recombinant protein or fragment.Preferably, refer to that those can combine with HGXPRT recombinant protein or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.The present invention also comprise those can with modify or without the HGXPRT recombinant protein bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the HGXPRT recombinant protein of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing HGXPRT recombinant protein or its has antigenic segmental cell and can be used to immune animal and produce antibody.Monoclonal antibody of the present invention can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Available HGXPRT recombinant protein of the production of polyclonal antibody or polypeptide immune animal, as rabbit, mouse, rat etc.Multiple adjuvant can be used for the enhancing immunity reaction, includes but not limited to freund adjuvant etc.
In another aspect of this invention, provide and contained recombinant protein of the present invention as the combinations of immunogens thing, especially vaccine composition, described composition contains the described recombinant protein of 0.001-99.9wt%, (b) pharmaceutically acceptable carrier and (c) optional adjuvant, wherein each component sum is 100wt%.Vaccine of the present invention can be preventative (being preventing infection) or curative (promptly treating disease after infection).
These vaccines comprise immunity antigen or immunogen, immunogenic polypeptide, albumen or protein fragments or nucleic acid (as Yeast Nucleic Acid or thymus nucleic acid), usually with " pharmaceutically acceptable carrier " combination, these carriers comprise itself does not induce any carrier of generation to the individual deleterious antibody of accepting said composition.Suitable carriers normally big, metabolism macromole slowly, as the virion of protein, polysaccharide, poly(lactic acid), polyglycolic acid, aminoacid polymers, amino acid copolymer, lipid agglutinator (as oil droplet or liposome) and non-activity.These carriers are well known to those of ordinary skill in the art.In addition, these carriers can play immunostimulant (" adjuvant ") effect.
The preferable adjuvant of enhancing composition effect is including, but not limited to (1) aluminium salt (alum), as aluminium hydroxide, aluminum phosphate, Tai-Ace S 150 etc.; (2) ISA720 adjuvant; (3) freund adjuvant etc.
Vaccine composition (comprising antigen, pharmaceutically acceptable carrier and/or adjuvant) contains thinner usually, as water, and salt solution, glycerine, ethanol etc.In addition, complementary material can be present in this class vehicle as wetting agent or emulsifying agent, pH buffer substance etc.In addition, the vaccine composition that comprises immunogenic composition can contain antigen, polypeptide, albumen, protein fragments or the nucleic acid in pharmaceutically acceptable carrier.
More specifically, comprise the vaccine of immunogenic composition, comprise the immunogenic polypeptide of immune significant quantity, and above-mentioned other required component." immune significant quantity " refers to that giving individual amount with a single agent or a continuous agent part is effective to treatment or prevention.This consumption according to the individual healthy state of treat and physiological situation, institute treat the ability of individual classification (as non-human primates etc.), individual immunity system synthetic antibody, required degree of protection, vaccine preparation, treat the doctor assessment of medical conditions, the correlative factor that reaches other decided.Estimate that this consumption will can determine by normal experiment in the scope of relative broad.
Usually, vaccine composition or immunogenic composition can be made the injectable agent, for example liquor or suspension; Also can be made into the solid form that before injection, is fit to allocate into solution or suspension, liquid excipient.But also emulsification or be encapsulated in the liposome of said preparation strengthens adjuvant effect under above-mentioned pharmaceutically acceptable carrier.
Ordinary method is to give immunogenic composition from parenteral (subcutaneous or intramuscular) approach by injection.That other prescription that is fit to other administering mode comprises is oral, suppository and transdermal application etc.Therapeutic dose can be single agent scheme or multi-agent scheme.Vaccine can give together in conjunction with other immunomodulator.
A kind of vaccine mode is a dna vaccination, promptly contains the dna vaccination of the encoding sequence of code book invention recombinant protein.
In an example of the present invention, complete synthesis malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase gene, and in pichia yeast this antigen of secreting, expressing, its conformation is very near native conformation by analysis.This antigenic immune serum can effectively suppress growth of malaria parasites, and has biological characteristicses such as ribosyl forwarding function.
Major advantage of the present invention is:
(1) HGXPRT recombinant protein of the present invention, its conformation be very near the native protein conformation, and have the ribosyl forwarding function.
(2) HGXPRT Recombinant Protein Expression product can be secreted into outside the born of the same parents, and enters no albumen culture supernatant, is convenient to separation and purification, and purity can reach more than 90%.
(3) HGXPRT Recombinant Protein Expression level height.Shaking bottle expression amount is 80mg/L, and 15L fermentation expression output can reach 1000mg/L.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989 or Chinese patent application CN01105292.9) described in condition, or the condition of advising according to manufacturer.
Synthesizing of Pfhgxprt gene
1.1.Pfhgxprt the design of complete synthesis gene
The proteic sequence of reorganization HGXPRT is from the aminoacid sequence of malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme HGXPRT.Select the pichia yeast codon usage frequency for use, this sequence is redesigned.Adopt analysis such as computer software " DNA Star ", Omiga to remove any sequence that may be unfavorable for genetic transcription and translation that in this gene order, exists side by side, as transcription termination sequence, intron splice site sequence, the inverted repeats grown etc.In addition, in 5 of newly-designed hgxprt gene ' and 3 ' end sets up XhoI and EcoRI restriction enzyme site respectively, so that can be connected with expression vector.Identify potential glycosylation site in this antigen sequence, and by glycosylation site amino acid Asn being transferred to Gln (seeing SEQ IDNO:2), thereby this glycosylation site got rid of.
MPIPNNPGAG?ENAFDPVFVK?DDDGYDLDSF?MIPAHYKKYL?TKVLVPNGVI 50
KNRIEKLAYD?IKKVYNNEEF?HILCLLKGSR?GFFTALLKHL?SRIHQYSAVE 100
MSKPLFGEHY?VRVKSYCNDQ?STGTLEIVSE?DLSCLKGKHV?LIVEDIIDTG 150
KTLVKFCEYL?KKFEIKTVAI?ACLFIKRTPL?WNGFKADFVG?FSIPDHFVVG 200
YSLDYNEIFR?DLDHCCLVND?EGKKKYKATS?LHHHHHH 237
(SEQ?ID?NO：2)
Add the partial sequence of yeast saccharomyces cerevisiae α-factor signal peptide leader sequence simultaneously at 5 of this gene ' end, the codon sequence (nucleotides sequence is classified aaa aga gag gct gaa gct as) that comprises signal peptide cutting identification amino acid Lys-Arg-Glu-Ala-Glu-Ala, make it secreting, expressing, and 3 ' end sets up the sequence of 6 Histidines of coding, so that use the Ni-NTA column purification.Consequent dna sequence dna is made up of 757bp, and names and be hgxprt.Like this, the HGXPRT recombinant protein is made up of 237 amino acid.
1.2.Pfhgxprt complete synthesis gene synthetic
Referring to Fig. 1.The Pfhgxprt synthetic gene sequence is divided into 8 oligonucleotide chains, from 5 ' to 3 ' successively called after Pfhgxprt-1 to Pfhgxprt-8, the lap of 16 to 20 bases of having an appointment between adjacent two oligonucleotide chains.Adopt gene software Oligo 4.0 to analyze adjacent oligonucleotide interchain lap sequences, be unfavorable for the sequence of oligonucleotide chain splicing, as hairpin structure etc. with eliminating.
With PCR method 8 chains are spliced into double chain DNA molecule.The pcr amplification condition is: 95 ℃ 10 seconds (sex change), 55 ℃ 30 seconds (annealing) and 72 ℃ 1 minute 30 seconds (extension), each 25 circulations produce 757bp total length Pfhgxprt synthetic gene (Fig. 2).
The PCR product separates with 1% sepharose, and reclaims test kit with QIAGEN DNA and separate goal gene.Cut target gene fragment with XhoI and the two enzyme enzymes of EcoRI, the enzyme tangent condition is: in 20 μ l reaction systems, contain 2 μ g target gene fragment, and 1 * enzyme cutting buffering liquid, each 5 unit of XhoI and EcoRI enzyme, 37 ℃ were reacted 1 hour.Endonuclease bamhi is connected with the pBluscript carrier of cutting through same enzyme, and transformed competence colibacillus DH5 α intestinal bacteria.The plasmid DNA of extracting acillin resistance bacterium colony is carried out dna sequence analysis after double digestion is accredited as correct insertion.Wrong base when synthesizing to get rid of.
The plasmid that will contain the faultless synthetic gene of total length changes bacillus coli DH 5 alpha over to, after inoculation culture repeatedly, reclaims this plasmid and carries out sequential analysis, and the result does not have the base mutation of discovery in the Pfhgxprt gene.Show in these synthetic gene energy stable existence intestinal bacteria.
For make the Pfhgxprt gene can be in pichia yeast secreting, expressing, this gene 5 ' end is modified, promptly add the partial sequence of yeast saccharomyces cerevisiae α-factor signal peptide leader sequence, comprise the codon sequence of signal peptide cutting identification amino acid Lys-Arg-Glu-Ala-Glu-Ala at 5 of this gene ' end.In 5 of Pfhgxprt gene ' and 3 ' end contains XhoI and EcoRI site respectively and is used for this gene clone and goes into expression vector.
Pfhgxprt gene secreting, expressing in pichia yeast
2.1: the structure of expression vector
The Pfhgxprt gene is inserted into pPIC9 expression plasmid of yeast (purchasing the company in lnvitrogen) by XhoI and EcoRI site.Like this, yeast saccharomyces cerevisiae α-factor signal peptide C-terminal merges on the N of PfHGXPRT end and this carrier.Because PfHGXPRT molecule N end contains three the special amino acid Glu-lys-Arg sequences in this signal peptide point of contact.So the target protein that secreting, expressing discharges does not contain signal peptide sequence.
The primary element of pPIC9K carrier is identical with pPIC9, but it has kalamycin resistance gene, and available G418 carries out the screening that high copy inserts son.Like this, will contain in the corresponding site that the Pfhgxprt fragment cut out and inserted pPIC9K expression plasmid (purchasing the company in lnvitrogen), be built into the pPIC9K/Pfhgxprt recombinant plasmid with BamHI and SalI.
2.2: transform pichia yeast SMD1168
With the pPIC9K/Pfhgxprt plasmid linearization, 10 μ g linearizing expression plasmids electricity transforms the SMD1168 pichia yeast with SalI.Transformed bacteria is used the transformant of different concns G418 plate screening multiple copied insertion then earlier with histidine defect plate screening His+ transformant.The genomic dna of the different transformants of extracting is identified with the inner primer PCR of a pair of goal gene, shows that expression plasmid inserts yeast chromosomal.There is the transformant of insertion to carry out following expression study through evaluation.
2.3:PfHGXPRT expression
It is in the substratum of carbon source that transformant is seeded in earlier with glycerine, grows after 24 hours, and being transferred to methyl alcohol is in the substratum of carbon source.Carry out abduction delivering.Cleer and peaceful cell precipitation on sampling in per 24 hours, the detection expression product.SDS-PAGE and Xylene Brilliant Cyanine G detected result show: tangible PfHGXPRT band of expression is arranged at the 26kD place, and this expression band only occurs behind methanol induction, and with the expression time prolongation, its expression product obviously increases (Fig. 3).
PfHGXPRT fermentation and purifying
Through optimization research, shake bottle expression level and can reach 80mg/L expression condition.Carry out fermentation expression with the 15L jar.In whole fermentation period, the cell in early stage is exponential growth and rises, and cell density reaches OD
600=110.Add vegetative period to glycerine, the cell growth is linearly risen, and cell density reaches OD
600=580.But arrived the methanol induction expression phase, the cell overall consistency remains unchanged substantially, and target protein to express be to add back 3-7hr at methyl alcohol to begin, and prolong in time, expressing productive rate improves rapidly, and constantly is secreted in the fermented liquid, high expression level amount can reach 1000mg/L.
The purifying of PfHGXPRT expression product divided for three steps carried out: the first step is to use the Ni-NTA column purification.Because the PfHGXPRT C-terminal contains 6 * His sequence, thus expression product can be attached on the Ni-NTA affinity column, and with 250mM imidazoles wash-out target protein.Second step was with gel-filtration liquid phase method purifying.The 3rd step was to carry out purifying with cation seperation column.Purity of protein through three step purifying can reach (Fig. 4) more than 90%.
The PfHGXPRT immune mouse
In this embodiment, be antigen with the PfHGXPRT of purifying, be the immunological adjuvant immune mouse with ISA720 and freund adjuvant respectively, experiment is divided into 5 groups, every group of 7 mouse, first group is ISA720+PfHGXPRT; Second group is freund adjuvant+PfHGXPRT; The 3rd group is ISA720 adjuvant control group; The 4th group is the freund adjuvant control group; The 5th group is the blank group.Immunity divides to be carried out for 3 times, and each immunity was carried out at interval in three weeks.Get hematometry specific antibody level through the tail vein before the immunity and about each immunity two weeks of back.Concrete grammar is as follows:
PfHGXPRT antigen dilutes with PBS, mixes by 3:7 with the ISA720 adjuvant, with syringe pump emulsification extremely evenly; After freund adjuvant was then pressed 1:1 and antigen is mixed, emulsification was not scattered to the emulsion droplets entry.Immunizing dose is every mouse 50 μ g, is 200 μ l volumes.Adopt subcutaneous multi-point injection.Three immunization times are respectively at W0, W3, W6.W2 and W4, W6 get hematometry antibody ELISA and IFA titre after first immunisation.
ELISA is undertaken by following program: adopting expression PfHGXPRT or the full worm albumen of plasmodium falciparum is antigen, every hole package amount is 0.1 μ g, add different dilution antiserum(antisera) 100 μ l, every part of dilute serum sample repeats three holes, after 37 ℃ of reaction 1hr and washing, add ELIAS secondary antibody,, and read 450nm OD value with the tmb substrate colour developing.Determine positive threshold value with preimmune serum, greater than the final antibody titers of the positive antiserum(antisera) of the extent of dilution of this value.
ELISA result shows that adjuvant and blank group do not produce specific antibody, and all the other 2 groups all produce tangible specific antibody (Fig. 5).The highest antibody titers is 1:111,080.
The above results shows that this recombinant protein demonstrates high immunogenicity in the mouse immune experiment.
The immune serum indirect immunofluorescence experiment
Immune serum indirect immunofluorescence experiment (IFA) is undertaken by following program: plasmodium falciparum (FCC1/HN strain) the blood membrane antigen sheet with cultivating, dry standby or-20 ℃ of preservations; The antigen sheet is separated with wax crayon; The immune mouse serum serial doubling dilution of 10mmol/L PBS (pH7.4) that contains 3% skim-milk.Drip 10 μ l dilute serums (the mouse-anti serum of preparation among the embodiment 4) in the middle of every antigen sheet cut zone, establish negative control simultaneously.Negative control is preimmune serum 1:200 dilution.37 ℃ of wet box 1h; PBS flushing three times; The concentration that adds the PBS dilution that contains 3% skim-milk in the part that drips immune serum is the FITC mark sheep anti-mouse igg of 0.01mg/ml, 37 ℃ of wet box 1h; PBS gives a baby a bath on the third day after its birth inferior, dries dark place, back fluorescence microscope.
IFA result as shown in Figure 6.Can produce the antibody of identification plasmodium falciparum native protein in the immunization experiment group mice serum, IFA result is positive; And control group and blank group IFA result are negative, and the highest IFA antibody titers of mice serum is 1:640.
Embodiment 6
The immanoprotection action of anti-challenge infection behind the PfHGXPRT immune mouse
Present embodiment uses five groups of mouse, and wherein the I group is ISA720+PfHGXPRT; The II group is freund adjuvant+PfHGXPRT; The III group is ISA720 adjuvant control group; The IV group is the freund adjuvant control group; The V group is the blank group.
Five groups of mouse the tenth day while abdominal injection 10 after immunity for the third time
5Individual blood passes Plasmodium yoelii (P.y) killer strain in 3 generations.Inoculation 10
5The compound method of the fresh blood of individual Plasmodium yoelii is:
Fresh blood liquid measure=10 of every mouse inoculation
5/ (the every μ l RBC number of mouse * protozoan infection rate)
Suitably dilute the fresh blood liquid measure of inoculation again with physiological saline, abdominal injection.Inoculate back 24 hours and rise, every day, regularly tail vein blood was made thin blood sheet, the dyeing of Ji Shi liquid, oily sem observation.Remember that plasmodium infects number in 12 visual field red corpuscle.
Five groups of mouse behind mouse Plasmodium yoelii challenge infection every 24 hours continuous microscopy 8 days, the person is decided to be the positive the parasitemia.
Behind the challenge infection 5 days, promptly mouse occurred before the death, infected the ratio of number and inspection mouse number again by the red corpuscle of daily inspection, obtained its average infection rate.Make figure with average infection rate.
The result such as the table 1 and shown in Figure 7 of immune attack test.
Table 1 Plasmodium yoelii (P.y) is attacked survival condition every day behind the mouse
The result shows: with respectively organizing average parasite rate behind the Plasmodium yoelii killer strain challenge infection on the 4th day there were significant differences (F check, P＜0.01), I group challenge infection after 24 hours 4 mouse parasitemia appears, parasitemia densities slowly rises, compared the red corpuscle infection rate behind the challenge infection in the 3rd day, the 4th day, the 5th day with III group, V group there were significant differences (T check, and all dead time retardations of mouse one day P＜0.05).II group challenge infection after 24 hours 6 mouse parasitemia appears, compare all death of section mouse at one time with the IV group, but parasitemia densities rises slowly, compared with IV group, V group in the 3rd day, the 4th day, the 5th day behind the challenge infection, (T check that there were significant differences for the red corpuscle infection rate, P＜0.05), whole death times of mouse are compared with the V group and postponed one day.It is similar to blank group that the adjuvant control group infects kinetics.
The above results proved, behind the PfHGXPRT immune mouse of the present invention, can provide the immanoprotection action of certain anti-challenge infection.
Embodiment 7
The ribosyl forwarding function of reorganization PfHGXPRT
HGPRT is active and the kinetic constant method for measuring is as follows: react total system 0.5ml, at 100 μ mol/L purine (when measuring guanine, concentration is 50 μ mol/L), 1mmoL/L PRPP, 100mmol/LTris-HCl pH7.4,12mmol/L MgCl
2In the damping fluid, add HGPRT, under 37 ℃, for xanthoglobulin, guanine, xanthine, VITAMIN B4 respectively 245,257,255,260nm measures the photoabsorption increase on PharmaciaBiotech Ultrospec2000 spectrophotometer.Corresponding △ ε is respectively 1900 (mol/L)
-1Cm
-1(xanthoglobulin-IMP), 5900 (mol/L)
-1Cm
-1(guanine .GMP), 3794 (mol/L)
-1Cm
-1(xanthine one XMP), 1600 (mol/L)
-1Cm
-1(VITAMIN B4 one AMP).At fixing 1mmol/L PRPP, changing purine concentration of substrate scope is under the condition of 5-100 μ mol/L, the initial velocity of assaying reaction, and calculate and obtain corresponding kinetic constant K
mAnd K
Cat
Enzyme dynamics and enzyme activity assay result show that reorganization HGXPRT has the reaction that catalysis ribose 5-phosphate tetra-sodium (PRPP) and xanthoglobulin or guanine produce purine nucleoside and inorganic pyrophosphate.This shows that reorganization HGXPRT has the biological characteristics of the ribosyl forwarding function identical with native protein.
Discuss
Malaria at present still serious threat human beings'health, the drug-fast generation of plasmodium and rapidly diffusion and anti-sterilant mosquito matchmaker appearance and spread, make the medical treatment of malaria be absorbed in predicament, so the effective malaria vaccine of development has become the task of top priority so that the control malaria is popular.The plasmodium complexity life history, antigen have kind, strain, phase specificity, and have serious antigenic variation, and this brings difficulty to vaccine development, thus the difficulty of malaria vaccine development than people imagined originally much higher.
In the life history, have only the erythrocytic stage protozoon to make the people pathogenic even fatal plasmodium, therefore develop sickness rate and the case fatality rate that effective erythrocytic stage malaria vaccine can reduce malaria, significant to the control of malaria.Immunoprotection mechanism for erythrocytic stage generally believes that specific antibody is critical factor; but also just causing widely, the function of cellular immunization pays close attention to; wherein studying maximum is the CD4+T cell. think about the functional point of view of CD4+T cell at present: it is except producing the antibody as the auxiliary B cell of Th cell, even direct action effect cell.
Result of study shows; malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl based transferase (PfHGXPRT) can be as the target antigen of protectiveness CD4+T cell; these T cells can pass to serious immunodeficient mouse (SCID mouse) with the ability of opposing plasmodium invasion, and this effect is that non-antibody relies on.Although the mechanism of action of these plasmodiums CD4+T cell special, that can suppress growth of malaria parasites is not illustrated at present as yet, a large amount of experimental evidences show that these CD4+T cells have the function that suppresses growth of malaria parasites really.Therefore, as the necessary complement of erythrocytic stage malaria vaccine development, be crucial for the research of the malaria vaccine candidate antigens that as PfHGXPRT, can excite the effective cell immunity.
Occurring in nature comprise plasmodium nearly 65000 surplus kind of protozoon all have unique metabolic characteristics, promptly all can not be by synthetic self the purine nucleotides of the approach of de novo synthesis purine ribonucleotide.In order to survive, plasmodium must pass through the purine salvage pathway.Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRT) plays an important role in the anabolic salvage pathway of plasmodium purine as elementary enzyme and the key enzyme of remedying purinase in the plasmodium body.Utilize the reaction mechanism of HGXPRT and biological characteristics can in preventing and treating parasitosis, bring into play vital role aspect following two: the expression product of the HGXPRT thing that serves as a mark; HGXPRT can be as the reasonable medicine target that participates in medicinal design.
The HGXPRT of plasmodium falciparum is the dimer or the tetramer, aminoacid sequence on the GenBank from be delivered to ncbi database as can be known, plasmodium falciparum HGXPRT albumen has the aminoacid sequence of high conservative, but HGXPRT of plasmodium falciparum and human HGPRT have very big difference on the specificity of substrate.To the research of the reaction mechanism of pernicious protozoacide HGXPRT, be to carry out from the angle of mixed potential electrons/molecule mechanism in the past.Utilize free enzyme simulation thing and hydridization current potential QM/MM to describe HGXPRT reaction mechanism in the plasmodium falciparum body, the back reaction path is the increase of shifting ratio along with glycosyl, limiting a fast proton transfers to the protein from xanthoglobulin, this is a stepping process, back single step reaction approach is the DNA transitory stage, in this stage, tetra-sodium separates the back quilt tightly in conjunction with living from group, rather than attack hypoxanthic nucleophilic group, and by testing the barrier value of having observed this enzyme.
In the HGXPRT of purification plasmodium falciparum process, the specific difference between it and the human HGPRT also is detected, and this helps to carry out rational medicinal design.The requirement of the purification condition of natural plasmodium falciparum HGXPRT is very strict, therefore comprises that by purifying natural PfHGXPRT the further research of vaccine development is extremely unpractical.
As the elementary enzyme of remedying purinase in the plasmodium body and the hypoxanthine-guanine-xanthine phosphoribosyl transferase of key enzyme, should be the rate-limiting enzyme in the plasmodium life Metabolic activity, just should be the plasmodium key link of the life history.For this reason, the inventor is through extensive and deep research, from the complete synthesis malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferase gene of dna level and express this recombinant protein.Found that this recombinant protein can cause immunne response effectively, and have excellent enzyme kinetics characteristic.Experimentation on animals also proves, can play the certain protection effect behind the reorganization HGXPRT protein immune animal, so the HGXPRT recombinant protein can be used as important candidate vaccine molecule.
Another practical problems of malaria vaccine is a human body MHC molecule polymorphism.Because the polymorphism of human body MHC molecule makes some antigens not combine with some body MHC molecule, causes antigen presentation to be obstructed, and the reactionless phenomenon of immune body occurs.For guaranteeing that vaccine obtains effective submission in most individualities, need in existing plasmodium antigens, to identify the many reactions epi-position that to discern majority MHC molecule, and mix vaccine, or a plurality of antigens are mixed recombinant antigen of composition to overcome monoclonal antibody former middle lack enough and MHC molecule bonded t cell epitope.
Existing research also shows, the plasmodium complexity life history, and antigen has the phase specificity.In addition, there is serious antigenic variation in plasmodium.Therefore, univalent vaccine be difficult to reach 100% efficient.Based on these characteristics, the malaria vaccine that effectively also can continue to use should comprise a plurality of plasmodium protective antigens, and consequent immunizing power can be carried out many-sided the attack.Like this, minority plasmodium thereby immune attack that escaped the first line of defence former owing to antigenic variation etc. survived, the attack in each defence line, road after these protozoons also can be subjected to is eliminated until all protozoons.The HGXPRT that relates among the present invention and the Pf ℃ of P-2.9 of malaria vaccine candidate antigens that enters the I clinical trial phase carry out the good experimental result that combined immunization obtained, for the exploitation polyvalent vaccine brings dawn.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Second Military Medical University, PLA
＜120〉recombinant malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyltransferase and method for making thereof and purposes
<130>054502
<160>2
<170>PatentIn?version?3.1
<210>1
<211>757
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(757)
＜223〉encoding sequence of HGXPRT recombinant protein
<220>
<221>CDS
<222>(25)..(735)
<223>
<400>1
<210>2
<211>237
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(757)
＜223〉encoding sequence of HGXPRT recombinant protein
<400>2
Claims (9)
1. recombinant protein, it is characterized in that, it comprises the aminoacid sequence of malignant malarial parasite hypoxanthine-guanine-xanthine phosphoribosyl transferring enzyme HGXPRT, and have the ribosyl forwarding function, the aminoacid sequence of described recombinant protein is shown in 1-231 position or 1-237 position among the SEQ ID NO:2.
2. an isolated DNA molecule is characterized in that, the described recombinant protein of its coding claim 1.
3. dna molecular as claimed in claim 2 is characterized in that, it is selected from down group:
(a) nucleotide sequence shown in the 25-735 position among the SEQ ID NO:1;
(b) nucleotide sequence shown in the 1-757 position among the SEQ ID NO:1.
4. a carrier is characterized in that, it contains the described dna molecular of claim 2.
5. a host cell is characterized in that, it contains the described carrier of claim 3.
6. a method that produces the described recombinant protein of claim 1 is characterized in that, comprises step:
Being fit to express under the condition of described recombinant protein, cultivate the described host cell of claim 5, thereby give expression to described recombinant protein;
Separate described recombinant protein.
7. method as claimed in claim 6 is characterized in that, the aminoacid sequence of described recombinant protein is shown in 1-237 position among the SEQ ID NO:2.
8. a composition is characterized in that, it contains the described recombinant protein of claim 1 or described dna molecular of claim 2 and pharmaceutically acceptable carrier.
9. composition as claimed in claim 8 is characterized in that described composition is a vaccine composition.
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