CN101955544A - Candida albicans secreted aspartic proteinase (Sap2) coding epitope sequence-synthesizing and bacteriophage shell protein-bonding hybrid protein and use thereof - Google Patents
Candida albicans secreted aspartic proteinase (Sap2) coding epitope sequence-synthesizing and bacteriophage shell protein-bonding hybrid protein and use thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of DNA recombination in bioengineering and particularly relates to a hybrid protein and use thereof. The hybrid protein can synthesize an epitope sequence for coding candida albicans secreted aspartic proteinase (Sap2) and can be bonded with the carrier of a phagemid. According to the analysis on the sequence of the bonded product, the coded protein sequence has 88 amino acids, and the molecular weight of the protein is 6.5kDa. The bonded carrier is used for expressing the hybrid protein in cell of Escherichia coli. The protein has high application values in detection and prevention of systemic Candida albicans infection.
Description
Technical field
The invention belongs to the DNA recombinant technology field in the biotechnology, be specifically related to hybrid protein and application thereof.
Background technology
Candida albicans is a kind of normal microflora that parasitizes health hosts skin, mucous membrane.When the immunity degradation of body or normal microflora balance are destroyed, can invade deep tissues and cause systemic candida albicans infection.It is a kind of yeast-like fungi, can exist with variforms such as spore, yeast and mycelium under different condition, and the antigen that different shape is expressed is also different.
Along with the use of tumor radiotherapy, chemotherapy and a large amount of Broad spectrum antibioticss, the sickness rate of systemic candida albicans infection constantly raises.Because candida albicans infection lacks tangible clinical symptom, so cause the early diagnosis difficulty.Clinically hemoculture methods that adopt more, this method need be through cultivating repeatedly, and length consuming time, recall rate are low, and just can obtain positive findings in the late period of infecting usually, and the patient often misses best treatment opportunity.Therefore, the early detection method of seeking systemic candida albicans infection is very important for the diagnosis and the treatment of such disease.Aspect treatment, mainly adopt antifungal drug treatment, antifungal drug not only price is expensive, and has very big side effect, as renal toxicity etc.Immunotherapy is considered to treat the effective way of fungi infestation.
Summary of the invention
The objective of the invention is to establish the hybrid protein of a kind of Candida albicans Sap2 epi-position and bacteriophage coat protein, and the application of this hybrid protein in the medicine of preparation system candida albicans infection clinical detection, prevention and treatment is provided.
The present invention adopts display technique of bacteriophage, and the antigen epitope genes of a kind of Candida albicans secretory proteolytic enzyme 2 (Sap2) is recombinated in a kind of expression plasmid that is integrated with the main coat protein gene of phage, forms recombinant vectors.Utilize CaCl
2Method is with recombinant vectors transformed into escherichia coli TG1 host cell.Foreign gene is at host cell inner expression, assembles and forms a kind of hybrid protein with bacteriophage coat protein, and be secreted into the extracellular with phage.Utilize biochemical method extraction and purifying to show the hybrid protein that epi-position is arranged.
Hybrid protein of the present invention is the filobactivirus gene 8 heterozygosis coat protein that contain Candida albicans Sap2 epi-position (SLAQVKYTSASSI).The nucleotides sequence of this hybrid protein gene is classified as:
atg?aaa?aag?tct?tta?gtc?ctc?aaa?gcc?tcc?gta?gcc?gtt?gct?acc?ctc 48
gtt?ccg?atg?ctg?tct?ttc?gcc?gcg?gag?ggt?tct?ttg?gct?caa?gtc?aaa 96
tat?act?tct?gct?tcc?agt?att?cca?tcg?aac?gat?cct?gca?aaa?gcg?gcc 144
ttt?gac?tcc?ctg?caa?gcc?tca?gcg?acc?gaa?tat?atc?ggt?tat?gcg?tgg 192
gcg?atg?gtt?gtt?gtc?att?gtc?ggc?gca?act?atc?ggt?atc?aag?ctg?ttt 240
aag?aaa?ttc?acc?tcg?aaa?gca?agc 264
The aminoacid sequence of this hybrid protein is:
Met?Lys?Lys?Ser?Leu?Val?Leu?Lys?Ala?Ser?Val?Ala?Val?Ala?Thr?Leu
1 5 10 15
Val?Pro?Met?Leu?Ser?Phe?Ala?Ala?Glu?Gly?Ser?Leu?Ala?Gln?Val?Lys
20 25 30
Tyr?Thr?Ser?Ala?Ser?Ser?Ile?Pro?Ser?Asn?Asp?Pro?Ala?Lys?Ala?Ala
35 40 45
Phe?Asp?Ser?Leu?Gln?Ala?Ser?Ala?Thr?Glu?Tyr?Ile?Gly?Tyr?Ala?Trp
50 55 60
Ala?Met?Val?Val?Val?Ile?Val?Gly?Ala?Thr?Ile?Gly?Ile?Lys?Leu?Phe
65 70 75 80
Lys?Lys?Phe?Thr?Ser?Lys?Ala?Ser
85
The preparation of this hybrid protein may further comprise the steps:
The first step, synthetic epitope gene
This gene is the gene of a kind of epi-position (SLAQVKYTSASSI) of coding Candida albicans Sap2.Synthesize with ordinary method by entrusting Shanghai to give birth to worker's biotechnology company limited.
In second step, make up recombinant vectors
Original plasmid is pfd88, handles original plasmid with two kinds of restriction enzymes, and the oligonucleotide fragment with synthetic is inserted into the plasmid vector of handling through enzyme then, and it is long to obtain recombinant vectors pfd8V.
The 3rd step is with the recombinant vectors transformed host cell.Host cell is the large intestine bacterial strain TG1 bacterial strain that has F-factor.The transformant that obtains is new engineering strain, and TG1V is long.
The 4th step, the preparation hybrid protein.The culturing engineering bacterial strain, steps such as process is centrifugal, precipitation can obtain the hybrid protein of this invention.
The hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein has the application of following aspect:
1. as the new biological product for preparing the detection system candida albicans infection
Enzyme linked immunosorbent detection shows that hybrid protein can be used as a kind of antigen and produces immunne response with the antibody of suffering from the systemic candida albicans infection person serum.Detecting positive rate reaches more than 90%.
2. as the new biological product of preparation treatment and prevention system candida albicans infection medicine, be used for the immunotherapy of systemic candida albicans infection.
Hybrid protein has produced humoral immunization and cellular immunization preferably at the mouse body:
(1) variation and the specificity of antibody in the mice serum of immune back have been detected with ELISA and Western blot method.The result shows: hybrid protein stimulates mouse to produce the specific antibody of anti-candida albicans Sap2 epi-position.
(2) the RT-PCR method detects mRNA level in the mice spleen of immunity back, and sandwich ELISA method detects the level of immune mouse spleen cell at external protein level secretion Th1 type and Th2 cytokines.The result shows that hybrid protein can produce the Th1 cytokines relevant with protection by the effective stimulus mouse boosting cell.
(3) hybrid protein shows better action in the immunoprophylaxis test of candida albicans infection mould mouse: experiment shows: immune hybrid protein has immanoprotection action to the candida albicans infection mouse; compare with negative control group; the infecting mouse kidney does not have inflammatory cell infiltration, and can improve the mortality ratio of infecting mouse.
Description of drawings
Accompanying drawing 1 is the electrophorogram of the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein;
Accompanying drawing 2 is hybrid protein mouse renal tissue section contrast figure in the immunoprophylaxis test of candida albicans infection mould mouse.
Embodiment
1. epitope gene is synthetic:
Utilize the dna synthesizer of commercial biotech firm to carry out the epitope gene fragment.The nucleotides sequence of this gene is classified as:
5’-tct?ttg?gct?caa?gtc?aaa?tat?act?tct?gct?tcc?agt?att-3’
2. the structure of recombinant vectors
Utilizing plasmid pfd88 is original plasmid, carries out the structure of recombinant vectors.
1) utilize restriction enzyme to handle original plasmid pfd88.
2) carrier after enzyme is cut mixes by mole concentration with the exogenous dna fragment of synthetic at 1: 3.
3) 10 μ L mixed solutions add 1 μ L T
4Dna ligase and 1 μ L damping fluid.Under 16 ℃ of conditions, spend the night, obtain recombinant vectors (pfd8V is long).
3. preparation competent cell
The TG1 cell cultures on the LB substratum, was cultivated 16-20 hour for 37 ℃.Select single colony inoculation in 2ml LB liquid culture medium, 37 ℃ of overnight incubation.Nutrient solution changes in the fresh LB nutrient solution of 100ml, cultivates 2.5 hours for 37 ℃.Centrifugal 10 minutes of 5000rpm collects thalline.0.1mol/L CaCl with the precooling of 20ml ice
2The suspension thalline, ice bath 10 minutes, centrifugal 10 minutes of 5000rpm.Remove supernatant, with the 0.1mol/L CaCl of 4ml ice precooling
2Resuspended thalline is preserved the competent cell packing of preparation, and every part adds 20% glycerine, and-70 ℃ of preservations are standby.
Put into 42 ℃ of water-baths 90 seconds, change in the ice bath 10 minutes then rapidly over to.0.1mol/L CaCl with the 8ml precooling
2Suspend and precipitate, obtain competent cell.
4. recombinant vectors transformed host cell
In connecting product, add the competent cell of 100 μ l, placed trash ice 30 minutes; Add 800 μ l LB nutrient solutions, place 37 ℃ of constant temperatures to cultivate 1 hour; Centrifugal 5 minutes of 10000rpm; Abandon the part supernatant, remainder is applied to the LB media surface that contains penbritin, overnight incubation in 37 ℃ of thermostat containers.Be selected in single bacterium colony of cultivating planar growth, carry out enlarged culturing and cryopreservation (70 ℃).
5. the preparation of hybrid protein
It is streak culture on the LB flat board to get frozen transformant, spends the night; The menu colony inoculation in the 3ml LB nutrient solution that contains penbritin, 37 ℃ of overnight incubation; Change nutrient solution in 100ml LB nutrient solution continuation cultivation, cultivated 2.5-4 minute for 37 ℃; Centrifugal 10 minutes; Precipitation suspends again with nutrient solution, cultivates a few hours for 37 ℃; Centrifugal 10 minutes, supernatant added solid polyethylene glycol and sodium-chlor, and 4 ℃ of placements are spent the night, and centrifugation suspends with TE liquid then; Supernatant adds 20% polyoxyethylene glycol and 2.5mol/L sodium-chlor behind the recentrifuge.Place a few hours, centrifugal 20 minutes for 4 ℃; Abandon supernatant, suspend with TE liquid and precipitate, obtain the filobactivirus heterozygosis coat protein that has allogenic polypeptide to show.
7. the detection of Zhi Bei hybrid protein
Adopt SDS-polyacrylamide gel electrophoresis to carry out proteic purity detecting
The testing protein sample was handled in boiling water 5 minutes.Carry out electrophoresis according to a conventional method.Behind the electrophoresis, offset plate is dyeed with silver nitrate method staining.If hybrid protein is expressed successfully, two bands appear on the offset plate.The top band is the hybrid protein band, and the below is a wild-type filamentous phage coat protein band.
8. immunological experiment
(1) antibody detection test
Utilize enzyme-linked immunoassay method, suffer from detection of antibodies in the systemic candida albicans infection person serum as antigen with hybrid protein.
Enzyme plate is antigen coated with every hole 3ug, after sealing, washing, adds systemic candida albicans infection patients serum, and ELIAS secondary antibody is used the goat anti-human igg of horseradish peroxidase-labeled, at last with TMB colour developing, sulfuric acid termination reaction.The result proves that hybrid protein can be by the specific antibody among patients serum identification, and does not contain this antibody in the normal human serum.
(2) humoral immunization of animal body and test for celluar immunity
Select 18-20 gram BALB/C mice random packet (experimental group and control group), 8 every group, carry out intraperitoneal injection (hybrid protein).The all immunity four times of each group, for the first time and immunity for the second time pitch time be 14 days, all the other immunity pitch times are 10 days, each immunity end of line portion blood sampling of advancing detects the variation of mouse internal antibody with the ELISA method.Result's proof is calculated according to OD＞2.1 of immune serum/control serum through after four immunity, and immune serum is tired and can be reached 200,000 times.
With the quadrat method immune mouse; go to mouse boosting tissue to carry out the RT-PCR detection respectively and prepare splenocyte suspension to carry out double antibodies sandwich ELISA detection; found that; hybrid protein can produce the Th1 cytokines relevant with protection by inducing mouse, relatively has significant difference (p＜0.05) with control group.
(3) animal immune prophylactic tria
Behind quadrat method usefulness hybrid protein immune mouse four times, tail intravenous inoculation 1 * 10
6Albicans cell.Got the infecting mouse kidney at postvaccinal the 7th day and carry out histological observation, carry out mortality ratio simultaneously and observe.Found that, in the control group mice renal plevis massive inflammatory cells infiltrated is arranged, and immune hybrid protein mouse kidney is normal, illustrates that hybrid protein has immanoprotection action to infecting mouse.
The nucleotide sequence of the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein:
atg?aaa?aag?tct?tta?gtc?ctc?aaa?gcc?tcc?gta?gcc?gtt?gct?acc?ctc 48
gtt?ccg?atg?ctg?tct?ttc?gcc?gcg?gag?ggt?tct?ttg?gct?caa?gtc?aaa 96
tat?act?tct?gct?tcc?agt?att?cca?tcg?aac?gat?cct?gca?aaa?gcg?gcc 144
ttt?gac?tcc?ctg?caa?gcc?tca?gcg?acc?gaa?tat?atc?ggt?tat?gcg?tgg 192
gcg?atg?gtt?gtt?gtc?att?gtc?ggc?gca?act?atc?ggt?atc?aag?ctg?ttt 240
aag?aaa?ttc?acc?tcg?aaa?gca?agc 264
The aminoacid sequence of the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein:
Met?Lys?Lys?Ser?Leu?Val?Leu?Lys?Ala?Ser?Val?Ala?Val?Ala?Thr?Leu
1 5 10 15
Val?Pro?Met?Leu?Ser?Phe?Ala?Ala?Glu?Gly?Ser?Leu?Ala?Gln?Val?Lys
20 25 30
Tyr?Thr?Ser?Ala?Ser?Ser?Ile?Pro?Ser?Asn?Asp?Pro?Ala?Lys?Ala?Ala
35 40 45
Phe?Asp?Ser?Leu?Gln?Ala?Ser?Ala?Thr?Glu?Tyr?Ile?Gly?Tyr?Ala?Trp
50 55 60
Ala?Met?Val?Val?Val?Ile?Val?Gly?Ala?Thr?Ile?Gly?Ile?Lys?Leu?Phe
65 70 75 80
Lys?Lys?Phe?Thr?Ser?Lys?Ala?Ser
85
Claims (3)
1. the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein, it is characterized in that Candida albicans Sap2 epi-position (SLAQVKYTSASSI) is showed in the main coat protein of filobactivirus surface, the hybrid protein that forms, this proteic amino acid structure sequence is:
Met?Lys?Lys?Ser?Leu?Val?Leu?Lys?Ala?Ser?Val?Ala?Val?Ala?Thr?Leu
1 5 10 15
Val?Pro?Met?Leu?Ser?Phe?Ala?Ala?Glu?Gly?Ser?Leu?Ala?Gln?Val?Lys
20 25 30
Tyr?Thr?Ser?Ala?Ser?Ser?Ile?Pro?Ser?Asn?Asp?Pro?Ala?Lys?Ala?Ala
35 40 45
Phe?Asp?Ser?Leu?Gln?Ala?Ser?Ala?Thr?Glu?Tyr?Ile?Gly?Tyr?Ala?Trp
50 55 60
Ala?Met?Val?Val?Val?Ile?Val?Gly?Ala?Thr?Ile?Gly?Ile?Lys?Leu?Phe
65 70 75 80
Lys?Lys?Phe?Thr?Ser?Lys?Ala?Ser
85；
The nucleotides sequence of this hybrid protein is classified as:
atg?aaa?aag?tct?tta?gtc?ctc?aaa?gcc?tcc?gta?gcc?gtt?gct?acc?ctc 48
gtt?ccg?atg?ctg?tct?ttc?gcc?gcg?gag?ggt?tct?ttg?gct?caa?gtc?aaa 96
tat?act?tct?gct?tcc?agt?att?cca?tcg?aac?gat?cct?gca?aaa?gcg?gcc 144
ttt?gac?tcc?ctg?caa?gcc?tca?gcg?acc?gaa?tat?atc?ggt?tat?gcg?tgg 192
gcg?atg?gtt?gtt?gtc?att?gtc?ggc?gca?act?atc?ggt?atc?aag?ctg?ttt 240
aag?aaa?ttc?acc?tcg?aaa?gca?agc 264。
2. the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein is as the application in the medicine of antibody in person's serum to be checked of preparation detection system candida albicans infection.
3. the hybrid protein of Candida albicans Sap2 epi-position and bacteriophage coat protein is as the application in the medicine of preparation treatment and prevention system candida albicans infection.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105467126A (en) * | 2014-09-09 | 2016-04-06 | 东北师范大学 | Phage composite nano-wires for detecting Candida albicans |
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|---|---|---|---|---|
| CN1680445A (en) * | 2005-01-12 | 2005-10-12 | 江西3L医用制品集团有限公司 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1680445A (en) * | 2005-01-12 | 2005-10-12 | 江西3L医用制品集团有限公司 | Antithemolytic streptococcus, staphylococcus and candida albicans lgG antibody and preparation thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《GenBank》 20000625 Smith,G.P et al AAF78532.1 1-3 , 2 * |
| 《中国优秀硕士学位论文》 20081115 张艳辉 肿瘤患者血清Hsp90和Sap2表位特异性抗体的Western blot检测 正文引言第5段 1-3 , 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105467126A (en) * | 2014-09-09 | 2016-04-06 | 东北师范大学 | Phage composite nano-wires for detecting Candida albicans |
| CN105467126B (en) * | 2014-09-09 | 2018-01-09 | 东北师范大学 | A kind of bacteriophage composite nano-line for being used to detect Candida albicans |
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Application publication date: 20110126 |