CN102212531A - Preparation method and application of recombinant Cladosporium herbarum allergen Cla h8 protein - Google Patents
Preparation method and application of recombinant Cladosporium herbarum allergen Cla h8 protein Download PDFInfo
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- CN102212531A CN102212531A CN2010101412091A CN201010141209A CN102212531A CN 102212531 A CN102212531 A CN 102212531A CN 2010101412091 A CN2010101412091 A CN 2010101412091A CN 201010141209 A CN201010141209 A CN 201010141209A CN 102212531 A CN102212531 A CN 102212531A
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Abstract
The invention belongs to the biological technical field and relates to a preparation method and an application of a recombinant Cladosporium herbarum allergen Cla h8 protein. RNAs are extracted from the cultured Cladosporium herbarum, the coding gene of the Cladosporium herbarum allergen Cla h8 protein is obtained through polymerase chain reaction (PCR) and the expression vector pET19b-Clah8 of the allergen Cla h8 protein is constructed. Expression purification and identification prove that the prepared recombinant Cladosporium herbarum allergen Cla h8 protein can be combined with the IgE and IgG in the serum of a patient allergic to Cladosporium herbarum in a specific manner and have the same immune activity with the natural Cladosporium herbarum allergen Cla h8 protein. The recombinant Cladosporium herbarum allergen Cla h8 protein with biological activity and solubility prepared by the method can avoid the problem that the non-singleness and standardization of the natural extract are difficult to realize. The prepared recombinant Cladosporium herbarum allergen Cla h8 protein can be used to prepare the preparation for diagnosing and curing the allergic diseases caused by Cladosporium herbarum.
Description
Technical field
The present invention relates to biological technical field, relate to the proteic preparation method and its usage of a kind of reorganization multi-trunk natalensis mould allergen Cla h8
Background technology
Allergic asthma is the serious public health problem of harm, and its sickness rate and mortality ratio are lasting ascendant trend.Estimate according to WHO, the asthmatic patient in the whole world existing 300,000,000, wherein adult more than 50% and at least 80% child patient bring out by irritated factor, have every year the patient more than 250,000 to die from asthma, asthma has been not only the public health problem of developed country, and nearly all country all deeply hurts.The statistics report, China mainland pathogenesis of asthma rate is still lower, is 2.1% (not comprising Hong Kong 6.2% and Taiwan 2.6%), but the mortality ratio of China's asthmatic patient occupies the whole world first up to 36.7/10 ten thousand.In addition, the difference of China's pathogenesis of asthma between each department can reach 10 times, and is the highest with Chongqing, Tibet is minimum, and in following 10 years, pathogenesis of asthma will continue to rise, because China's large population base, the pathogenesis of asthma rate rises 2 percentage points, with the number of the infected that increases more than 2,000 ten thousand.
Studies show that allergic asthma can be brought out by multiple factor, the various allergens that wherein exist in the environment are its main induced factors.These allergens comprise scurf, pollen and the mould etc. of dirt mite, animal.In warm and humid environment, mould is the main inducing that causes anaphylactic disease.It is reported, the south in the U.S., 30% allergy patient is to multiple mould sensitivity.Multi-trunk natalensis mould (Cladosporium herbarum) is exactly a kind of mould that mainly causes anaphylactic disease, and it is not only a kind of main indoor anaphylactogen, also is the main outdoor anaphylactogen in two seasons of autumn in summer.
Multi-trunk natalensis is mould to contain multiple allergen protein, and wherein Cla h8 allergen protein is its main allergen protein.IgE antibody in the mould allergy patient's serum of 57% above multi-trunk natalensis can specific identification Cla h8 allergen protein.The skin prick experimental result shows that Cla h8 can cause that the redness of skin appears in the patient.
What clinically the mould allergy patient's of multi-trunk natalensis diagnosis and specific antigens immunotherapy are adopted at present is traditional mould leach liquor of multi-trunk natalensis, it contains multiple antigenic component, and there are evident difference in relative potency and specific antigens level, it is quantitative to be difficult to carry out stdn, cause that easily diagnosis or therapeutic anaphylaxis appear in the patient, therefore limited its diagnosis and treatment are used.And the mould allergen protein of the multi-trunk natalensis output height not only of reorganization, working condition is constant, helps carrying out stdn, is more suitable for carrying out clinical diagnosis and treatment.
Summary of the invention
The objective of the invention is to carry out deficiency in the Clinics and Practices of anaphylactic disease, a kind of reorganization multi-trunk natalensis mould allergen Cla h8 is provided proteic preparation method and its usage at the mould allergen leach liquor of multi-trunk natalensis.
The present invention passes through genetic engineering means, from the mould tissue of multi-trunk natalensis, extract the encoding gene of its main allergen protein Cla h8, construction of expression vector, the mould allergen PROTEIN C of abduction delivering reorganization multi-trunk natalensis la h8 in intestinal bacteria, the mould allergen PROTEIN C of the reorganization multi-trunk natalensis la h8 that the present invention makes can be used for the Clinics and Practices of the mould anaphylactic disease that causes of multi-trunk natalensis.
Particularly, the invention provides the expression method of the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8, it is characterized in that it comprises the steps:
(1) from the mould thalline of cultivating of multi-trunk natalensis, extracts total RNA, by the synthetic cDNA of RT-PCR;
(2) synthetic primer:
Cla?h8-S?5-CCCATATGCCTGGCCAGCAAGCAA-3
Cla?h8-as?5-CGGGATCCTTATCTGGTGGTGTAACCA-3
(3) with cDNA be template, obtain the mould allergen PROTEIN C of multi-trunk natalensis la h8 encoding gene fragment by PCR; Described Cla h8 encoding gene is shown in SEQ ID No:1, and its coded aminoacid sequence is shown in SEQ ID No:2;
(4) encoding gene is cloned in the expression vector, transforms the host bacterium, cultivate the gained recombinant bacterial strain, induce it to express target protein then;
(5) adopt damping fluid that rCla h8 inclusion body is washed, after it is purified, adopt the gsh restoring system, thereby obtain the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8 its renaturation;
(6) immunological method is identified the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8 immunogenicity.
Among the present invention, described expression vector is not limited to specific expression vector, if it can with described cDNA gene recombination, form suitable expression plasmid.Preferred expression carrier of the present invention is a prokaryotic expression carrier; The present invention more preferably prokaryotic expression carrier is pET19b.
Among the present invention, described host cell is not limited to any specific host cell, as long as it can express described recombinant expression vector.The preferred host bacterium of the present invention is e. coli bl21 Star
TM(DE3) pLysS, substratum are the LB liquid nutrient medium, and culture temperature is 20～37 ℃, incubation time 6～16 hours.
Among the present invention, the described inductor of step (5) is IPTG.The thalline of cultivating carries out abduction delivering with IPTG after reaching suitable concentration, and inductive condition is: IPTG concentration is 0.1～1mM, and inducing temperature is 20～37 ℃, and induction time is 2～16 hours.
Among the present invention, the described purification process of step (6) is: with the mould allergen PROTEIN C of multi-trunk natalensis la h8 express engineering bacteria 37 ℃ induce 3 hours after, centrifugal collection thalline places ultrasonic degradation on ice, adds 30 ℃ of joltings of N,O-Diacetylmuramidase 30 minutes then, centrifugal, collection inclusion body protein precipitation, with the refolding kit of Novagen company to inclusion body protein purify with renaturation after, centrifugal 20 minutes of 14000rmp, collect supernatant, refrigerator is preserved.
Among the present invention, adopt Dolt bolt and Western blot method to detect the immunogenicity of rCla h8.
Among the present invention, the expression vector of employing, host cell are commercial.
The present invention has following advantage compared with prior art:
The present invention extracts total RNA from the multi-trunk natalensis of cultivating is mould, amplify the encoding gene of the mould main allergen protein Cla h8 of multi-trunk natalensis, thus construction of expression vector.After changing intestinal bacteria over to and inducing, the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8 efficiently expresses in intestinal bacteria with the inclusion body form, after simple washing, can obtain highly purified albumen.After with the gsh restoring system it being carried out renaturation, can obtain to have the mould allergen PROTEIN C of the solubility reorganization multi-trunk natalensis la h8 of biologic activity, can be used for the diagnosis and the treatment of the mould anaphylactic disease that causes of multi-trunk natalensis, the present invention has avoided the non-singularity of natural extract and the obstacle of stdn difficulty.
Description of drawings
Fig. 1, the mould allergen Cla of pcr amplification multi-trunk natalensis h8 protein coding gene electrophorogram.
Fig. 2, the Hind III single endonuclease digestion of the mould allergen Cla of multi-trunk natalensis h8 protein expression vector pET19b-cla h8 is identified electrophorogram.
Fig. 3, the BamHI of the mould allergen Cla of multi-trunk natalensis h8 protein expression vector pETl9b-cla h8 and NdeI double digestion are identified electrophorogram.
Fig. 4, the mould allergen Cla of multi-trunk natalensis h8 protein coding gene and the contrast of AY191816 sequence.
Fig. 5, the contrast of the mould allergen Clah8 of multi-trunk natalensis Argine Monohydrochloride sequence.
Fig. 6, the mould allergen Cla of reorganization multi-trunk natalensis h8 albumen detects the SDS-PAGE electrophoresis result at expression in escherichia coli,
Wherein, M is low molecular protein marker, and 1 is inductive bacterial lysate not, and 2 for after inducing 3 hours through 37 ℃ of the IPTG of 1mM, bacterial lysate.
Fig. 7, the mould allergen Cla of reorganization multi-trunk natalensis h8 albumen inclusion body protein SDS-PAGE detects electrophoresis result behind the purifying.
Fig. 8 recombinates, and SDS-PAGE detects electrophoresis result after the reconstruct of the mould allergen Cla of multi-trunk natalensis h8 albumen inclusion body protein renaturation.
Fig. 9, the mould allergen Cla of reorganization multi-trunk natalensis h8 proteantigen specificity Dolt blotting detects
Wherein, 1 is CBB dyeing, and 2 are and the mould allergic disease human serum IgG of multi-trunk natalensis reaction result, and 3 is the mould allergic disease human serum of multi-trunk natalensis IgE reaction result.
Figure 10, the mould allergen Cla of reorganization multi-trunk natalensis h8 egg western blot detects,
Wherein, M is low molecular weight protein (LMWP) marker, and 1 for the mould allergen Cla of reorganization multi-trunk natalensis h8 albumen dyes through CBB, and 2,3,4 are respectively IgE reaction result in reorganization multi-trunk natalensis mould allergen Cla h8 albumen and the mould allergic disease human serum of multi-trunk natalensis.
Specific embodiments
The mould cultivation of embodiment 1 multi-trunk natalensis
Multi-trunk natalensis mould (Cladosporium herbarum) bacterial strain of preserving (ATCC 6056) is inoculated in 100ml YPD substratum (10g/L yeast extract, 20g/L peptone, 20g/L glucose), pH6.0～6.5), cultivated 6 days for 25～28 ℃, the centrifugal supernatant that goes, PBS damping fluid washing thalline three times ,-80 ℃ of preservations are standby.
The mould allergen Cla of embodiment 2 multi-trunk natalensis h8 encoding gene obtains
According to the operation instruction of Rneasy Total RNA test kit, extract total RNA of the mould thalline of multi-trunk natalensis, use GeneAmp RNA PCR Kit to carry out RT-PCR, obtain cDNA.The mRNA sequence (AY191816) of the mould allergen Cla of the multi-trunk natalensis h8 that announces according to GeneBank, the design primer also adds NdeI and the BamHI restriction enzyme site: Cla h8-S 5-CCCATATGCCTGGCCAGCAAGCAA-3 and Clah8-as 5-CGGGATCCTTATCTGGTGGTGTAACCA-3 are template with cDNA, the gene fragment of the mould allergen Cla of pcr amplification coding multi-trunk natalensis h8 mature peptide section, the PCR product detects through 2% agarose gel electrophoresis, and its size is 804bp (Fig. 1).
The structure of embodiment 3 recombinant expression plasmid pET19b-Cla h8
With QIAquick PCR Purification Kit purification pcr amplification product, cut pcr amplification product with NdeI and BamHI enzyme again and insert in the pET19b carrier.Change e. coli jm109 over to, bacterium liquid is coated the LB agar plate (contain the blue or green 100 μ g/ml of ammonia benzyl, paraxin 34 μ g/ml), after 37 ℃ of incubated overnight, the picking clone, the extracting recombinant plasmid carries out double digestion to be identified, the display-object band is consistent with the PCR product size of Cla h8 gene fragment as a result, the recombinant plasmid pET19b-Cla h8 (Fig. 2 that has successfully made up the mould allergen Cla of multi-trunk natalensis h8 is described, Fig. 3) by the handsome biological company limited in Shanghai to the evaluation of checking order of this recombinant plasmid, and adopt Vector NTI 10 softwares, putative amino acid sequence, and with Genebank in the sequence (AY191816) announced compare.The nucleotide sequence of rClah8 lacks the presequence (being made up of 51 Nucleotide) and the C-terminal non-coding region (being made up of 63 Nucleotide) of N-terminal, and has 9 nucleotide sites different with AY191816, but do not cause amino-acid residue change (Fig. 4, Fig. 5).
The proteic expression of embodiment 4 reorganization multi-trunk natalensis mould allergen Cla h8
Behind recombinant plasmid pET19b-Cla h8 transformed into escherichia coli BL21Star (DE3) pLysS, coat LB agar plate (containing the blue or green 100 μ g/ml of ammonia benzyl, paraxin 34 μ g/ml), overnight incubation in 37 ℃ of incubators.Select single bacterium colony and go in the 800ml LB nutrient solution (containing the blue or green 50 μ g/ml of ammonia benzyl, paraxin 34 μ g/ml) 37 ℃ of 220rmp joltings, be cultured to OD600 and be about at 0.5 o'clock, add the IPTG of 1mM, induced 3 hours for 37 ℃, centrifugal 10 minutes of 4 ℃ of 8000rpm abandon supernatant, collect bacterial precipitation., the SDS-PAGE through 12.5% detects, and IPTG inductive bacterium liquid target protein band of expression (Fig. 6) occurs at the about 37kD of molecular weight place.
The mould allergen Cla of embodiment 5 reorganization multi-trunk natalensis h8 is proteic folding again
The mould allergen Cla of multi-trunk natalensis of the present invention h8 is with the formal representation of inclusion body.In the bacterial precipitation, add 40ml IB wash buffer and 40 μ l 1M PMSF, ultrasonic on ice after, add N,O-Diacetylmuramidase (final concentration is 200 μ g/ml) mixing after, 30 ℃ of 100rmp joltings are after 30 minutes, and are ultrasonic on ice once more, centrifugal 10000rmp10 minute; Abandon supernatant, after adding 40ml IB wash buffer suspended again, centrifugal 10 minutes of 10000rmp repeated this step, until sedimentary solid colour.SDS-PAGE through 12.5% detects, and the proteic inclusion body purity of the mould allergen Cla of multi-trunk natalensis h8 can reach (Fig. 7) more than 98%.
Change in the 50ml centrifuge tube of a known weight inclusion body after suspending over to 10000rmp centrifugal 20 minutes; Abandon supernatant, and clean liquid remaining on the wall, after the weighing, add an amount of IB solubilizationbuffer, making its final concentration is 20mg/ml; Blow and beat repeatedly after inclusion body protein all dissolves with suction pipe, room temperature left standstill 15 minutes; Centrifugal 10 minutes of room temperature 10000rmp; Its supernatant is changed in the dialysis tubing, dialyse.
Dialysis tubing is put into the Dialysis buffer that contains 0.1mM DTT, and 4 ℃ of dialysis repeated this dialysis procedure once after 3 hours; Change the Dialysis buffer do not contain DTT, 4 ℃ of dialysis are after 3 hours, change Dialysis buffer after, 4 ℃ of dialysed overnight; Dialysis tubing is put into Redox refolding buffer, continue 4 ℃ of dialysis more than 8 hours, after room temperature was dialysed 1 hour again, 4 ℃ of refrigerators were preserved.SDS-PAGE through 12.5% detects, and the mould allergen Clah8 of the multi-trunk natalensis of renaturation reconstruct molecular weight of albumen is 37KDa, and purity can reach (Fig. 8) more than 98%.
The mensuration of the mould allergen Cla of embodiment 6 reorganization multi-trunk natalensis h8 protein content
Use DC Protein Assay test kit to detect recombinant protein content.Bovine serum albumin (BSA) is a standard protein, with PBS prepare respectively different concns BSA reference liquid (1.5mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml 0mg/ml), sets up BSA protein concentration typical curve.According to typical curve, calculating recombinant protein concentration is 1.8mg/ml.Every liter of bacterium can prepare the mould allergen Cla of 22.5mg multi-trunk natalensis h8 albumen.
The mould allergen Cla of embodiment 7 reorganization multi-trunk natalensis h8 proteantigen specificity Dolt blotting detects
Respectively with the mould extractive substance 2 μ g of multi-trunk natalensis and reorganization multi-trunk natalensis mould allergen Cla h8 albumen 2 μ g application of samples on nitrocellulose filter, after the drying at room temperature, add the PBS that contains 3%BSA, the room temperature sealing is 1 hour in the wet box; Confining liquid is abandoned in suction, adds respectively the mould asthma patient serum hypersensitive of multi-trunk natalensis (dilution in 1: 2 and dilution in 1: 100), 4 ℃ of overnight incubation; PBST washes film, 30 minutes; Add HRP-Mouse Anti-Human IgE antibody (dilution in 1: 250) and HRP-Mouse Anti-Human IgG antibody (dilution in 1: 250) respectively, incubated at room 1 hour; PBST washes film, and 30 minutes, with Konica Immunostaining HRP-100 colour developing, termination reaction behind the 20min.Dolt blotting result shows that IgE antibody and the IgG antibody in the mould asthma patient serum hypersensitive of multi-trunk natalensis can combine with mould extractive substance of multi-trunk natalensis and reorganization multi-trunk natalensis mould allergen Cla h8 albumen generation specificity, illustrates that the mould allergen Clah8 of reorganization multi-trunk natalensis albumen has identical immunocompetence (Fig. 9) with native protein.
The mould allergen Cla of embodiment 8 reorganization multi-trunk natalensis h8 proteantigen specificity Western blotting detects
Get the mould allergen Cla of 6 μ g multi-trunk natalensis h8 albumen, add equal-volume sample buffer, 1/10 volume beta-mercaptoethanol, 95 ℃ of sex change are 5 minutes behind the mixing, and centrifugal 10 minutes of 14000rpm, supernatant are used for SDS-PAGE and go up sample.Electrophoresis forwards the albumen electricity on the pvdf membrane to after finishing.After electricity changeed end, PBST washed film after 30 minutes, added the PBS that contains 5% skimmed milk, and the room temperature sealing is 1 hour in the wet box; Confining liquid is abandoned in suction, adds the mould asthma patient serum hypersensitive of multi-trunk natalensis (dilution in 1: 2), 4 ℃ of overnight incubation; PBST washes film, 30 minutes; Add HRP-Mouse Anti-Human IgE antibody (dilution in 1: 250), incubated at room 1 hour; PBST washes film, and 30 minutes, with Konica Immunostaining HRP-100 colour developing, termination reaction behind the 20min.Westernblot result shows that 3 all with the mould allergen Clah8 of multi-trunk natalensis albumen in various degree reaction, wherein No. 2 patient reaction's property the strongest (Figure 10) take place to the IgE antibody in the mould asthma patient serum hypersensitive of multi-trunk natalensis.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉preparation method and its usage of the mould allergen PROTEIN C of a kind of multi-trunk natalensis of recombinating la h8
<130>
<160>2
<170>PatentIn?version?3.5
<210>1
<211>804
<212>DNA
＜213〉multi-trunk natalensis mould (Cladosporium herbarum)
<220>
<221>CDS
<222>(1)..(804)
<400>1
atg?cct?ggc?cag?caa?gca?acc?aag?cat?gag?tcc?ctt?ttg?gac?cag?ctc 48
Met?Pro?Gly?Gln?Gln?Ala?Thr?Lys?His?Glu?Ser?Leu?Leu?Asp?Gln?Leu
1 5 10 15
tcc?ctg?aag?ggc?aag?gtc?gtc?gtc?gtc?acc?ggc?gct?tcc?ggc?ccc?aag 96
Ser?Leu?Lys?Gly?Lys?Val?Val?Val?Val?Thr?Gly?Ala?Ser?Gly?Pro?Lys
20 25 30
ggc?atg?ggt?att?gag?gcc?gct?cgc?ggc?tgc?gcc?gag?atg?ggc?gcc?gct 144
Gly?Met?Gly?Ile?Glu?Ala?Ala?Arg?Gly?Cys?Ala?Glu?Met?Gly?Ala?Ala
35 40 45
gtt?gcc?atc?acc?tac?gct?tcc?cgc?gcc?cag?ggt?gct?gag?gag?aac?gtc 192
Val?Ala?Ile?Thr?Tyr?Ala?Ser?Arg?Ala?Gln?Gly?Ala?Glu?Glu?Asn?Val
50 55 60
aag?gag?ctc?gag?aag?acc?tac?ggc?atc?aag?gcc?aag?gcc?tac?aag?tgc
240
Lys?Glu?Leu?Glu?Lys?Thr?Tyr?Gly?Ile?Lys?Ala?Lys?Ala?Tyr?Lys?Cys
65 70 75 80
cag?gtc?gac?agc?tac?gag?tcc?tgt?gag?aag?ctc?gtc?aag?gac?gtc?gtt 288
Gln?Val?Asp?Ser?Tyr?Glu?Ser?Cys?Glu?Lys?Leu?Val?Lys?Asp?Val?Val
85 90 95
gcc?gac?ttc?ggc?cag?atc?gat?gcc?ttt?att?gcc?aac?gcc?ggt?gcc?acc 336
Ala?Asp?Phe?Gly?Gln?Ile?Asp?Ala?Phe?Ile?Ala?Asn?Ala?Gly?Ala?Thr
100 105 110
gcc?gac?tct?ggc?atc?ctc?gac?ggc?tcc?gtc?gag?gcc?tgg?aac?cac?gtc 384
Ala?Asp?Ser?Gly?Ile?Leu?Asp?Gly?Ser?Val?Glu?Ala?Trp?Asn?His?Val
115 120 125
gtc?cag?gtc?gac?ctg?aac?ggt?acc?ttc?cac?tgc?gcc?aag?gca?gtt?ggc 432
Val?Gln?Val?Asp?Leu?Asn?Gly?Thr?Phe?His?Cys?Ala?Lys?Ala?Val?Gly
130 135 140
cac?cac?ttc?aag?gag?cgt?gga?acc?ggt?tcc?ctc?gtc?atc?acc?gct?tcc 480
His?His?Phe?Lys?Glu?Arg?Gly?Thr?Gly?Ser?Leu?Val?Ile?Thr?Ala?Ser
145 150 155 160
atg?tcc?ggc?cac?atc?gcc?aac?ttc?ccc?cag?gag?cag?acc?tcc?tac?aac 528
Met?Ser?Gly?His?Ile?Ala?Asn?Phe?Pro?Gln?Glu?Gln?Thr?Ser?Tyr?Asn
165 170 175
gtc?gcc?aag?gct?ggc?tgc?atc?cac?atg?gct?cgc?tcc?ctc?gcc?aac?gag 576
Val?Ala?Lys?Ala?Gly?Cys?Ile?His?Met?Ala?Arg?Ser?Leu?Ala?Asn?Glu
180 185 190
tgg?cgc?gac?ttc?gcc?cgt?gtc?aac?tcc?atc?tcc?ccc?ggt?tac?att?gac 624
Trp?Arg?Asp?Phe?Ala?Arg?Val?Asn?Ser?Ile?Ser?Pro?Gly?Tyr?Ile?Asp
195 200 205
act?ggt?ctc?tcc?gac?ttc?gtc?ccc?aag?gag?acc?cag?cag?ctc?tgg?cac 672
Thr?Gly?Leu?Ser?Asp?Phe?Val?Pro?Lys?Glu?Thr?Gln?Gln?Leu?Trp?His
210 215 220
tcc?atg?atc?ccc?atg?ggc?cgt?gac?ggt?ctc?gcc?aag?gag?ctc?aag?ggc 720
Ser?Met?Ile?Pro?Met?Gly?Arg?Asp?Gly?Leu?Ala?Lys?Glu?Leu?Lys?Gly
225 230 235 240
gcc?tac?gtc?tac?ttc?gcc?tcc?gac?gcc?tcc?acc?tac?acc?acc?ggt?gcc 768
Ala?Tyr?Val?Tyr?Phe?Ala?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ala
245 250 255
gat?ctc?ctc?att?gac?ggt?ggt?tac?acc?acc?aga?taa 804
Asp?Leu?Leu?Ile?Asp?Gly?Gly?Tyr?Thr?Thr?Arg
260 265
<210>2
<211>267
<212>PRT
＜213〉multi-trunk natalensis mould (Cladosporium herbarum)
<400>2
Met?Pro?Gly?Gln?Gln?Ala?Thr?Lys?His?Glu?Ser?Leu?Leu?Asp?Gln?Leu
1 5 10 15
Ser?Leu?Lys?Gly?Lys?Val?Val?Val?Val?Thr?Gly?Ala?Ser?Gly?Pro?Lys
20 25 30
Gly?Met?Gly?Ile?Glu?Ala?Ala?Arg?Gly?Cys?Ala?Glu?Met?Gly?Ala?Ala
35 40 45
Val?Ala?Ile?Thr?Tyr?Ala?Ser?Arg?Ala?Gln?Gly?Ala?Glu?Glu?Asn?Val
50 55 60
Lys?Glu?Leu?Glu?Lys?Thr?Tyr?Gly?Ile?Lys?Ala?Lys?Ala?Tyr?Lys?Cys
65 70 75 80
Gln?Val?Asp?Ser?Tyr?Glu?Ser?Cys?Glu?Lys?Leu?Val?Lys?Asp?Val?Val
85 90 95
Ala?Asp?Phe?Gly?Gln?Ile?Asp?Ala?Phe?Ile?Ala?Asn?Ala?Gly?Ala?Thr
100 105 110
Ala?Asp?Ser?Gly?Ile?Leu?Asp?Gly?Ser?Val?Glu?Ala?Trp?Asn?His?Val
115 120 125
Val?Gln?Val?Asp?Leu?Asn?Gly?Thr?Phe?His?Cys?Ala?Lys?Ala?Val?Gly
130 135 140
His?His?Phe?Lys?Glu?Arg?Gly?Thr?Gly?Ser?Leu?Val?Ile?Thr?Ala?Ser
145 150 155 160
Met?Ser?Gly?His?Ile?Ala?Asn?Phe?Pro?Gln?Glu?Gln?Thr?Ser?Tyr?Asn
165 170 175
Val?Ala?Lys?Ala?Gly?Cys?Ile?His?Met?Ala?Arg?Ser?Leu?Ala?Asn?Glu
180 185 190
Trp?Arg?Asp?Phe?Ala?Arg?Val?Asn?Ser?Ile?Ser?Pro?Gly?Tyr?Ile?Asp
195 200 205
Thr?Gly?Leu?Ser?Asp?Phe?Val?Pro?Lys?Glu?Thr?Gln?Gln?Leu?Trp?His
210 215 220
Ser?Met?Ile?Pro?Met?Gly?Arg?Asp?Gly?Leu?Ala?Lys?Glu?Leu?Lys?Gly
225 230 235 240
Ala?Tyr?Val?Tyr?Phe?Ala?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ala
245 250 255
Asp?Leu?Leu?Ile?Asp?Gly?Gly?Tyr?Thr?Thr?Arg
260 265
Claims (11)
1. the preparation method of the mould allergen PROTEIN C of the multi-trunk natalensis of recombinating a la h8 is characterized in that it comprises the steps:
1) from the mould thalline of cultivating of multi-trunk natalensis, extracts total RNA, by the synthetic cDNA of RT-PCR;
2) synthetic primer:
Cla?h8-S?5-CCCATATGCCTGGCCAGCAAGCAA-3
Cla?h8-as?5-CGGGATCCTTATCTGGTGGTGTAACCA-3
3) with cDNA be template, obtain the mould allergen PROTEIN C of multi-trunk natalensis la h8 encoding gene fragment by PCR;
4) encoding gene is cloned in the expression vector, transforms the host bacterium, cultivate the gained recombinant bacterial strain, induce it to express target protein then;
5) adopt damping fluid that rCla h8 inclusion body is washed, after it is purified, adopt the gsh restoring system, obtain the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8 its renaturation;
6) immunological method is identified the mould allergen PROTEIN C of reorganization multi-trunk natalensis la h8 immunogenicity.
2. method according to claim 1 is characterized in that, in the described step 3), Cla h8 encoding gene is shown in SEQID No:1; Coded aminoacid sequence is shown in SEQ ID No:2.
3. method according to claim 1 is characterized in that the expression vector of described step 4) is not limited to specific expression vector, if it can with described cDNA gene recombination, form suitable expression plasmid.
4. according to claim 1 or 3 described methods, it is characterized in that described expression vector is a prokaryotic expression carrier.
5. according to claim 1 or 3 described methods, it is characterized in that described expression vector is pET19b.
6. method according to claim 1 is characterized in that, the host bacterium of described step 4) is not limited to any specific host cell, as long as it can express described recombinant expression vector.
7. method according to claim 6, wherein said host cell are e. coli bl21 Star
TM(DE3) pLysS.
8. method according to claim 1 is characterized in that, in the described step 4), the substratum of host bacterium is the LB liquid nutrient medium, and culture temperature is 20～37 ℃, incubation time 6～16 hours.
9. method according to claim 1 is characterized in that, in the described step 4), thalline is used the IPTG abduction delivering after reaching suitable concentration, and inductive condition is: IPTG concentration is 0.1～1mM, and inducing temperature is 20～37 ℃, and induction time is 2～16 hours.
10. method according to claim 1, it is characterized in that the purification process in the described step 5) is: with the mould allergen PROTEIN C of multi-trunk natalensis la h8 express engineering bacteria 37 ℃ induce 3 hours after, centrifugal collection thalline, place ultrasonic degradation on ice, add 30 ℃ of joltings of N,O-Diacetylmuramidase 30 minutes then, centrifugal, collect the inclusion body protein precipitation, with the refolding kit of Novagen company to inclusion body protein purify with renaturation after, centrifugal 20 minutes of 14000rmp collects supernatant, and refrigerator is preserved.
11. the purposes of the mould allergen PROTEIN C of the reorganization multi-trunk natalensis la h8 that the method for claim 1 makes in the mould anaphylactic disease preparation that causes of preparation Clinics and Practices multi-trunk natalensis.
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| US9850281B2 (en) | 2012-06-01 | 2017-12-26 | Circassia Limited | Cladosporium peptides |
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