CN1207307C - Recombination human cytomegalovirus fusion protein and its preparing method, application - Google Patents
Recombination human cytomegalovirus fusion protein and its preparing method, application Download PDFInfo
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Abstract
The present invention discloses a recombination human cytomegalovirus fusion protein and a preparation method thereof, which relates to the fields of a genetic engineering technique and a diagnostic reagent. In the present invention, the genetic engineering technique is used for preparing a novel recombination protein, namely the fusion protein formed by the serial connection of 25 amino acids at the C end of the PP150 protein of a human cytomegalovirus and 164 amino acids at the C end of the gp 52 protein. The 25 amino acids at the C end of the PP150 protein are at the N end of the fusion protein, the 164 amino acids at the C end of the gp 52 protein are at the C end of the fusion protein, and the 25 amino acids at the C end of the PP150 protein and the 164 amino acids at the C end of the gp 52 protein are connected by the two amino acids of gly and ser, wherein the 162th amino acid in the 164 amino acids at the C end of the gp 52 protein is replaced with asn by lysine, one met is added at the N end of the fusion protein, and the full length of the fusion protein is equal to the length of 192 amino acids. The protein is used for detecting human cytomegalovirus antibodies or antigens and preparing monoclonal antibodies and polyclone antibodies.
Description
Technical field
The present invention utilizes genetic engineering technique, preparation recombinant human cytomegalovirus fusion protein.Two fragments of fusion rotein come from the gp52 albumen and the PP150 albumen of human cytomegalic inclusion disease virus respectively, two fragments are utilized genetic engineering technique to connect and are expressed, form new fusion rotein, the new fusion protein of generation can be used as antigen, is used for human cytomegalic inclusion disease virus antibody or detection of antigens etc.The present invention relates to genetic engineering technique and diagnostic reagent field.
Technical background
Human cytomegalic inclusion disease virus (Human cytomegalovirus writes a Chinese character in simplified form HCMV) is a kind of dna virus, and it can be hidden after infecting human body in vivo for a long time, and the generation recurrent infection that can be activated.The pregnant woman that HCMV infects both can pass through the placental infection fetus, can infect to the newborn infant through birth canal and breast milk again, can produce grievous injury [Rawlinson WD.Broadsheet.Pathology, 1999 such as microcephalus, dysnoesia behind fetus and the infection of newborn HCMV; 31 (2): 109～115].Acquired immune deficiency syndrome (AIDS) and other immunodeficiency type patient infection HCMV are one of major reason that causes death [Rawlinson WD.Broadsheet.Pathology, 1999; 31 (2): 109～115].In addition, often find also that in the patient of organ transplantation HCMV infects.
Set up the method for quick of HCMV pregnant woman and blood donor etc. is checked, propagate significant prenatal and postnatal care and prevention HCMV.The method that detects HCMV at present mainly comprises: the cell cultures of HCMV virus, and the PCR method detects HCMV DNA, and ELISA method and immunoblotting detect HCMV antibody (IgM and IgG) and antigen.Wherein the ELISA method detects method [Daininer A, Bader U, Eggers M, et al.J Clin Virol, 1999 that HCMV antibody or antigen are the most normal employings; 13 (3): 161～171].
Multiple HCMV viral protein can stimulate body to produce corresponding antibody.Should select the proteantigen that antigenicity is strong, specificity is good for use, be used to detect HCMV antibody.Mostly the HCMV viral protein antigen that China uses is to cultivate viral extract, contains a plurality of protein products, and antigen-specific is not strong, easily produces false positive.External [Vornhagen R, Plachter B, Hinderer W, et al.J Clin Microbiol, 1994 of studies confirm that; 32:981～985.PlachterB, Weiczorek l, Ziegelmaier B, et al.J Clin Microbiol, 1992; 30:201～206], gp52 albumen and the PP150 albumen of HCMV have stronger antigenicity, its corresponding antibody recall rate is higher in HCMV the infected's serum, but make Detection of antigen HCMV antibody with the gp52 of total length and PP150 albumen and false positive can occur, therefore, should select for use the part fragment that its endoantigen is strong, specificity is good to make antigen.
One section gp52 protein fragments that we once will screen and 25 amino acid amalgamation and expressions of PP150 PROTEIN C end [Li Yuexi, Tao Kaihua, Wang Xingtai, etc.The amalgamation and expression of human cytomegalic inclusion disease virus PP150 PROTEIN C end polypeptide and gp52 protein fragments. Chinese microbiology and Journal of Immunology, 2001; 9:21 (5): 556～559], purifying has obtained highly purified fusion rotein, and the fusion rotein of preparation has antigenicity and specificity preferably.But make Detection of antigen HCMV IgM antibody positive serum with this fusion rotein, it is more shallow to develop the color when finding test section serum, and pushing away then is because the gp52 protein fragments position of selecting is not too accurate, has influenced the formation of epitope.
Summary of the invention
The present invention utilizes genetic engineering technique to prepare a kind of novel recombinant protein, the fusion rotein that to be human cytomegalic inclusion disease virus PP150 PROTEIN C end form from 433 amino acid whose 164 amino acid series connection of the 270th amino acid to the from 1048 amino acid whose 25 amino acid of the 1024th amino acid to the and gp52 PROTEIN C end, 25 amino acid of PP150 PROTEIN C end are held at fusion rotein N, 164 amino acid of gp52 PROTEIN C end are at the C of fusion rotein end, be connected by 2 amino acid of glycine (Gly) and Serine (Ser) between the two, wherein the 162nd amino acid in 164 amino acid of gp52 PROTEIN C end replaces with asparagus fern acyl acid (Asn) by Methionin (Lys), the N end of fusion rotein has increased by 1 egg base acid (Met), 192 amino acid of total length.This albumen is used to detect human cytomegalic inclusion disease virus antibody or antigen and is used for the immune anti-human cytomegalic inclusion disease virus monoclonal antibody and how anti-for preparing.
Recombinant human cytomegalovirus fusion protein and preparation method thereof, application take following steps to implement:
HCMV PP150 PROTEIN C end group synthesizes and renaturation because of segmental:
Utilize softwares such as ANTHEWIN, by the proteic aminoacid sequence (GeneBank of Computer Analysis HCMV PP150, closing number: M16022), 25 amino acid finding the proteic C end of HCMV PP150 can form stronger antigenic determinant, and 25 amino acid are: Gly Gly Ala Lys Thr Pro Ser Asp Ala Val Gln Asn Ile LeuGln Lys Ile Glu Lys Ile Lys Asn Thr Glu Glu.Select these amino acid whose bacterium preference codons for use, synthetic two complementary dna fragmentations, normal chain is: 5 '-CATGGGC GGT GCT AAA ACT CCG TCT GAC GCTGTT CAG AAC ATC CTG CAG AAA ATC GAA AAA ATC AAA AAC ACT GAA GAA G-3 '; Minus strand is: 5 '-GATCC TTC TTC AGT GTT TTT GAT TTT TTC GAT TTT CTG CAG GAT GTT CTG AACAGC GTC AGA CGG AGT TTT AGC ACC GCC-3 '.Article two, 5 ' of chain end has all carried out phosphorylation.Article two, form above-mentioned 25 amino acid whose gene fragments after chain annealing and the renaturation, and form a Nco I sticky end at 5 ' end, form a BamHI sticky end at 3 ' end, make this gene fragment be easy to be cloned in plasmid pET28a (+) interior the Nco I and BamHI restriction enzyme site.
The screening of HCMV gp52 PROTEIN C end epitope and the clone of gene fragment:
1) screening of epitope
Utilize softwares such as ANTHEWIN, by the proteic whole aminoacid sequences of Computer Analysis HCMV gp52 (GeneBank, closing number: X17403), filter out the strong antigen epi-position in the gp52 albumen, promptly from 433 amino acid of the 270th amino acid to the, its aminoacid sequence and dna sequence dna are as follows:
Epitope aminoacid sequence in the screening gp52 (270aa～433aa):
Glu?Pro?Phe?Gln?Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser?Gly?Lys
Ser?Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn?Ala?Gly?Gly
Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn?Glu?Pro?Met?Gly?Leu
Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Lys?Lys?His?Asp?Arg?Gly?Gly?Gly
Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met?Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp?His?Asp?His
Gly?Leu?Ser?Ser?Lys?Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu?Thr?Ser
Lys?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn?Ser
Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp?Ser?Val?Thr?Phe
Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Lys?Cys?Gly
The dna sequence dna of the proteantigen epi-position of screening (808～1302bp):
GAA?CCT?TTC?CAG?CGT?GGC?GAT?CCC?TTC?GAC?AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC?AAG
TCG?CGT?GGC?GGC?GGC?GGT?GGT?GGC?GGC?AGC?CTC?TCT?TCG?CTG?GCC?AAT?GCC?GGC?GGT
CTG?CAT?GAC?GAC?GGC?CCG?GGT?CTG?GAT?AAC?GAT?CTC?ATG?AAC?GAG?CCC?ATG?GGT?CTC
GGC?GGT?CTG?GGA?GGA?GGT?GGC?GGC?GGT?GGC?GGC?AAG?AAG?CAC?GAC?CGC?GGT?GGC?GGC
GGT?GGT?TCC?GGT?ACG?CGG?AAA?ATG?AGC?AGC?GGT?GGC?GGC?GGC?GGT?GAT?CAC?GAC?CAC
GGT?CTT?TCC?TCC?AAG?GAA?AAA?TAC?GAG?CAG?CAC?AAG?ATC?ACC?AGC?TAC?CTG?ACG?TCC
AAA?GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA?GGA?GGA?GGC?GGC?GGT?TTG?GAT?CGC?AAC?TCC
GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG?GAG?AGC?GAC?AGC?GAG?GAT?TCT?GTA?ACG?TTC
GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA?AAG?TGC?GGC?TAA
2) gene clone of gp52 PROTEIN C end epitope
Select design PCR primer in the both sides of the dna sequence dna of above-mentioned epitope.The upstream primer of design is P1 (808-825bp): 5 '-CGGGATCCGAACCTTTCCAGCGTGGC-3 '; Downstream primer is P2 (1285-1302bp): 5 '-CGAATTCTTAGCCGCAGTTTTGCTTCTT-3 '.On upstream primer, increased BamH I restriction enzyme site (following setting-out part), EcoR I restriction enzyme site (following setting-out part) and whole subcipher on downstream primer, have been increased, in addition, sequence to downstream primer during synthetic primer has been carried out rite-directed mutagenesis, change bases G (italic) into by original base C, this codon amino acids coding becomes Asn (N) by Lys (K).
Get HCMV (the AD169 strain is provided by Chinese pharmaceutical biological product) cells and supernatant 200 μ l, add 100 μ l Proteinase Ks (10mg/ml), put 56 ℃ of water-baths 4 hours.With phenol/chloroform method purifying HCMV DNA, the HCMV DNA that obtains is dissolved in the 20 μ l deionized waters, put-20C is frozen standby.
HCMV DNA with above-mentioned purification is a template, and with the gene fragment of primer P1, P2 pcr amplification gp52 PROTEIN C end epitope, amplification condition is: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations; Last 72 ℃ were extended 7 minutes.Electrophoresis reclaims, the gene fragment of purifying amplification, is dissolved in the deionized water, put-20 ℃ frozen standby.
The expressed fusion protein construction of recombinant plasmid:
Extract plasmid pET28a (+) (U.S. Novagen company product),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the deionized water with Nco I and EcoR I double digestion.The positive and negative chain of gene fragment of synthetic PP150 C end polypeptide is dissolved in respectively in the damping fluid (50mmol/L Tris-HCl pH7.4,50mmol/L NaCl), and the final concentration that dissolves back two chains is identical.Get the positive and negative chain solution of equivalent and place mixing in the same centrifuge tube, put 100 ℃ of water-soluble interior sex change 5min, take out rearmounted room temperature naturally cooling, making two chain renaturation is double-stranded gene fragment, its 5 ' formation Nco I sticky end, and 3 ' end forms BamH I sticky end.
With the gp52 gene fragment with EcoR I and BamH I double digestion purifying, electrophoresis is dissolved in the deionized water after reclaiming.
Above-mentioned three kinds of enzymes of volumetric molar concentration such as get and cut the back dna fragmentation, in same centrifuge tube, connect with the T4 dna ligase, PP150 PROTEIN C end group is connected with the gp52 gene fragment because of fragment to be inserted between carrier pET28a (+) interior the Nco I and EcoR I site, both translate the framework unanimity, express a fusion rotein.
The screening of recombinant plasmid and evaluation:
With recombinant plasmid transformed e. coli bl21 (DE3), coating contains kantlex (60 μ g/ml) LB flat board, puts 37 ℃ and spends the night.Next day, picking transformed a bacterium colony and a contrast bacterium (plasmid pET28a transformed bacteria) at random, extracted plasmid respectively.At first use Nde I restriction analysis, in a Nde I of plasmid pET28a (+) internal memory restriction enzyme site (between Nco I and BamH I site), after foreign gene inserts between Nco I and the BamH I site, NdeI restriction enzyme site between two sites is removed, therefore the plasmid after the reorganization can not be cut by Nde I enzyme, but not the plasmid of reorganization then can be cut by Nde I.The plasmid that will contain foreign gene carries out dna sequence analysis, and sequential analysis confirms that recombinant plasmid contains placed in-line PP150 PROTEIN C end group because of fragment and gp52 gene fragment, and sequence is entirely true:
ATG?GGC?GGT?GCT?AAA?ACT?CCG?TCT?GAC?GCT?GTT?CAG?AAC?ATC?CTG?CAG?AAA?ATC
GAA?AAA?ATC?AAA?AAC?ACT?GAA?GAA?GGA?TCC?GAA?CCT?TTC?CAG?CGT?GGC?GAT?CCC?TTC
GAC?AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC?AAG?TCG?CGT?GGC?GGC?GGC?GGT?GGT?GGC?GGC
AGC?CTC?TCT?TCG?CTG?GCC?AAT?GCC?GGC?GGT?CTG?CAT?GAC?GAC?GGC?CCG?GGT?CTG?GAT
AAC?GAT?CTC?ATG?AAC?GAG?CCC?ATG?GGT?CTC?GGC?GGT?CTG?GGA?GGA?GGT?GGC?GGC?GGT
GGC?GGC?AAG?AAG?CAC?GAC?CGC?GGT?GGC?GGC?GGT?GGT?TCC?GGT?ACG?CGG?AAA?ATG?AGC
AGC?GGT?GGC?GGC?GGC?GGT?GAT?CAC?GAC?CAC?GGT?CTT?TCC?TCC?AAG?GAA?AAA?TAC?GAG
CAG?CAC?AAG?ATC?ACC?AGC?TAC?CTG?ACG?TCC?AAA?GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA
GGA?GGA?GGC?GGC?GGT?TTG?GAT?CGC?AAC?TCC?GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG
GAG?AGC?GAC?AGC?GAG?GAT?TCT?GTA?ACG?TTC?GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA
AAC?TGC?GGC?TAA
The expression of recombinant plasmid PP150 PROTEIN C end (25 amino acid) that makes up and the fusion rotein of gp52 PROTEIN C end (164 amino acid), be connected by 2 amino acid of glycine (Gly) and Serine (Ser) between the two, wherein the 162nd amino acid in 164 amino acid of gp52 PROTEIN C end replaces with asparagus fern acyl acid (Asn) by Methionin (Lys), the N end of fusion rotein has increased egg base acid (Met), 192 amino acid of total length, its aminoacid sequence is as follows:
Met?Gly?Gly?Ala?Lys?Thr?Pro?Ser?Asp?Ala?Val?Gln?Asn?Ile?Leu?Gln
5 10 15
Lys?Ile?Glu?Lys?Ile?Lys?Asn?Thr?Glu?Glu?Gly?Ser?Glu?Pro?Phe?Gln
20 25 30
Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser?Gly?Lys?Ser
35 40 45
Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn?Ala
50 55 60
Gly?Gly?Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn
65 70 75 80
Glu?Pro?Met?Gly?Leu?Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly
85 90 95
Lys?Lys?His?Asp?Arg?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met
100 105 110
Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp?His?Asp?His?Gly?Leu?Ser?Ser?Lys
115 120 125
Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu?Thr?Ser?Lys?Gly
130 135 140
Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn
145 150 155 160
Ser?Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp
165 170 175
Ser?Val?Thr?Phe?Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Asn?Cys?Gly
180 185 190
The Screening and Identification of expressed fusion protein engineering bacteria:
The positive transformant that will contain recombinant plasmid, be seeded to and contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h, add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects, and recon is expressed relative molecular weight and is about 22000 fusion rotein, expression amount is about 35%, and contrast bacterium BL21 (DE3) does not have this protein band.
The purifying of expressed fusion protein:
1) ultrasonic degradation of expressed fusion protein engineering bacteria
Centrifugal (8000rpm, 10min, 4 ℃) receives bacterium with the engineering bacteria of abduction delivering fusion rotein, thalline is resuspended in the lysate (50mmol/L Tris-HCl pH8.15,10mmol/L EDTA, 10mmol/L DTT) of original fluid 1/10 volume, ice-bath ultrasonic is broken bacterium 10min, centrifugal (12000rpm, 20min, 4 ℃) collects supernatant, abandons precipitation.The supernatant of collecting is used for next step ion-exchange purification.
2) DEAE-Sepharose FF anion column purifying
DEAE-Sepharose FF anion column is connected to the normal pressure chromatographic system, earlier with balance liquid (50mmol/LTris-HCl pH8.15,1mmol/L EDTA, 0.1mmol/L DTT) flushing balance, to go up the cracking supernatant of step acquisition then and directly go up sample, last sample flow velocity is 1.5ml/min.Behind the last sample with balance liquid flushing, then successively with contain 50,100, the balance liquid eluted protein of 1000mmol/L NaCl, collect 100mmol/L NaCl elution peak albumen, this peak contains the fusion rotein of expression.
3) S-Sepearse FF cation seperation column purifying
The elution peak that contains fusion rotein (100mmol/LNaCl peak) with last step DEAE-Sepharose FF negatively charged ion purifying obtains in the dialysis tubing of packing into, stirs dialysis 4h to dialyzate (20mmol/L PB (pH7.4), 1mmol/L EDTA, 0.1mmol/LDTT).With above-mentioned dialyzate flushing balance S-Sepharose FF cation seperation column, the fusion rotein after will dialysing is then directly gone up sample, last sample flow velocity 1.5ml/min.Fully wash post with dialyzate behind the end of the sample, more successively with contain 50,100, the dialyzate eluted protein of 1000mmol/L NaCl, collect 100mmol/L NaCl elution peak albumen, be the fusion rotein of purifying.
The fusion rotein of purifying is used as Detection of antigen HCMV antibody:
Wrap by elisa plate behind fusion rotein usefulness carbonate (pH9.6) doubling dilution of 50mmol/L with purifying, the indirect enzyme-linked immunosorbent method detects known anti-gp52 IgM (or IgG) positive serum and anti-PP150 IgM (or IgG) positive serum and normal human serum, the result shows, fusion rotein can react with anti-gp52 IgM (or IgG) and anti-PP150IgM (or IgG) positive serum, not with the normal human serum reaction, illustrate that interior gp52 protein fragments of this fusion rotein and PP150 PROTEIN C end protein all have better antigenicity and specificity.
Same this fusion rotein of horseradish peroxidase (HRP) mark of using detects anti-HCMV IgM or IgG antibody with pouncing on the method for obtaining, and higher susceptibility and specificity are arranged.This fusion rotein detects anti-HCMV IgM or IgG antibody as antigen with golden mark method, and higher susceptibility and specificity are also arranged.
The advantage that the present invention compared with prior art has
Mostly the HCMV viral protein antigen of China's use at present is to cultivate viral extract, contains a plurality of protein products, and antigen-specific is not strong, easily produces false positive.External HCMV gp52 albumen and the PP150 albumen that uses has stronger antigenicity, and its corresponding antibody recall rate is higher in HCMV the infected's serum, false positive can occur but make Detection of antigen HCMV antibody with the gp52 of total length and PP150 albumen.Therefore, should select for use the part fragment that its endoantigen is strong, specificity is good to make antigen.
HCMV PP150 PROTEIN C end polypeptide and gp52 PROTEIN C end fragment fusion rotein that we express have more advantage:
When 1. making the anti-HCMV-IgM serum of Detection of antigen, no longer need to prepare respectively this two kinds of albumen, also do not need to adjust two antigenic usage ratios with these two albumen, therefore use convenient and cost lower.
2. these two proteic antigenicities are stronger fragment amalgamation and expression, not only antigenicity is strong, and can reduce non-specific responding, simultaneously, has also avoided little peptide to make antigenic instability problem.
3. when detecting anti-HCMV IgM with prize law, only need this a kind of fusion rotein of enzyme labelling, no longer need two albumen of mark respectively.Therefore, when detection antibody needed a plurality of proteantigen, preparation multivalence fusion rotein antigen was one of ideal developing direction.
4. the expression HCMV PP150 C of this paper structure holds the engineering bacteria of the fusion rotein of polypeptide and gp52 C end, and expression amount can reach 35% of thalline soluble proteins, and exists with soluble form, is easy to purifying, but large-scale purification prepares this fusion rotein.Do not see Table at present the report that reaches this fusion rotein.
5. we once held HCMV PP150 C another section protein fragments (from 420 amino acid of the 255th amino acid to the) amalgamation and expression of polypeptide and gp52 C end, but, a little less than the antigenicity of the fusion rotein that its antigenicity is applied for a patent than this paper, so it is not applied for a patent.
Description of drawings
The invention will be further described below with reference to accompanying drawing:
Fig. 1 carries out analytical results with multiple molecular biology software to the epitope in the HCMV gp52 albumen.The result shows, contains strong wetting ability epitope at the proteic C end of gp52 from 420 amino acid of the 255th amino acid to the, i.e. the position that arrow indicates in the figure.
Fig. 2 is the construction of recombinant plasmid schema of expressing HCMV PP150 C end polypeptide and gp52 C end fusion rotein.
Fig. 3 is the pcr amplification product with 5 recons of Agarose gel detection of 1.2%.M: the low-molecular-weight dna standard (TaKaRa, DL2000); C: the contrast bacterium that contains plasmid pET28a (+); 1～5:5 transformant all amplifies the target gene fragment of 588bp, i.e. the position that arrow indicates in the figure.
Fig. 4 is the SDS-PAGE analytical results of expressing PP150 C end polypeptide and gp52 C end fusion rotein reorganization bacterium.1～5:1～No. 5 reorganization bacterium, 5 recons are all expressed relative molecular weight and are about 22000 fusion rotein, i.e. the position that arrow indicates in the figure; 6: contrast bacterium E.coli BL21 (DE3).
Fig. 5 is the SDS-PAGE analytical results of expressing gp52 protein purification front and back.M: low molecular weight protein (LMWP) standard (Pharmacia); 1: contrast bacterium BL21 (DE3); 2: the engineering bacteria of expressing PP150 and gp52 fusion rotein; 3: engineering bacteria ultrasonic degradation supernatant; 4: with the PP150 and the gp52 fusion rotein of DEAE-Sepharose FF anion column purifying; Behind the 5:S-Sepharose FF cation seperation column purifying PP150 and gp52 fusion rotein.
Fig. 6 is PP150 and gp52 fusion rotein antigen immune trace result.1: normal human serum; 2: anti--HCMVgp52 protein I gM positive human serum; 3: anti--HCMV PP150 protein I gM positive human serum.Orange precipitation band shown in the arrow.
Embodiment
The detailed description of embodiment of the present invention:
25 amino acid of human cytomegalic inclusion disease virus PP150 PROTEIN C end and gp52 albumen
C holds 164 amino acid whose amalgamation and expressions
Select the codon of bacterium preference for use, 25 amino acid whose gene fragments of chemosynthesis PP150 PROTEIN C end.With PCR method amplification gp52 protein gene fragment.To same plasmid pET28a (+) interior NcoI/BamHI and BamHI/EcoRI site, both are cascaded these two gene fragment clones, and translation framework unanimity, a fusion rotein can be expressed.With recombinant plasmid transformed e. coli bl21 (DE3), screening has obtained to efficiently express the engineering bacteria of this fusion rotein, and the fusion rotein of expression accounts for about 35% of tropina total amount, and exists with soluble form.
Materials and methods
1. bacterial classification and plasmid: host bacterium BL21 (DE3) and expression vector pET28a (+) are U.S. Novagen company product.
2. molecular biology reagent: restriction enzyme NcoI, NdeI, BamHI, EcoRI, and the T4 dna ligase be TaKaRa company product.Plasmid purification test kit and the test kit that reclaims dna fragmentation in the sepharose are German QIAGEN company product.DTT and IPTG are Promega company product.Other reagent is import or homemade analytical reagent.
3. gene fragment is synthetic: helped synthetic by Dalian TaKaRa company.
The enzyme of gene clone method: DNA cut, connection, electrophoresis; The extraction of plasmid, conversion; General molecular cloning methods such as proteic SDS-PAGE analysis carry out according to a conventional method.Other test kit by specification is operated.
5.DNA sequential analysis: with QIAGEN company plasmid purification test kit plasmid purification, with the full-automatic sequenator order-checking of DNA.
The result
1.HCMV PP150 PROTEIN C end group synthesizes and renaturation because of segmental:
Utilize softwares such as ANTHEWIN, by the proteic aminoacid sequence (GeneBank of Computer Analysis HCMV PP150, closing number: M16022), 25 amino acid finding the proteic C end of HCMV PP150 can form stronger antigenic determinant, and 25 amino acid are: Gly Gly Ala Lys Thr Pro Ser Asp Ala Val Gln Asn Ile LeuGln Lys Ile Glu Lys Ile Lys Asn Thr Glu Glu.
Select these amino acid whose bacterium preference codons for use, synthetic two complementary dna fragmentations, normal chain is: 5 '-CATGGGC GGT GCT AAA ACT CCG TCT GAC GCT GTT CAG AAC ATC CTG CAG AAA ATCGAA AAA ATC AAA AAC ACT GAA GAA G-3 '; Minus strand is: 5 '-GATCC TTC TTC AGT GTT TTT GATTTT TTC GAT TTT CTG CAG GAT GTT CTG AAC AGC GTC AGA CGG AGT TTT AGC ACC GCC-3 '.Article two, 5 ' of chain end has all carried out phosphorylation.Article two, form above-mentioned 25 amino acid whose gene fragments after chain annealing and the renaturation, and form a Nco I sticky end at 5 ' end, form a BamHI sticky end at 3 ' end, make this gene fragment be easy to be cloned in plasmid pET28a (+) interior the Nco I and BamHI restriction enzyme site.
2.gp52 the screening and the gene clone of PROTEIN C end epitope:
Utilize softwares such as ANTHEWIN, by the proteic whole aminoacid sequences of Computer Analysis HCMV gp52 (GeneBank, closing number: X17403), filter out the strong antigen epi-position in the gp52 albumen, promptly from 433 amino acid of the 270th amino acid to the, its aminoacid sequence and dna sequence dna are as follows:
Epitope aminoacid sequence in the screening gp52 (270aa～433aa):
Glu?Pro?Phe?Gln?Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser?Gly?Lys
Ser?Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn?Ala?Gly?Gly
Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn?Glu?Pro?Met?Gly?Leu
Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Lys?Lys?His?Asp?Arg?Gly?Gly?Gly
Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met?Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp?His?Asp?His
Gly?Leu?Ser?Ser?Lys?Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu?Thr?Ser
Lys?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn?Ser
Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp?Ser?Val?Thr?Phe
Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Lys?Cys?Gly
The dna sequence dna of the proteantigen epi-position of screening (808～1302bp):
GAA?CCT?TTC?CAG?CGT?GGC?GAT?CCC?TTC?GAC?AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC?AAG
TCG?CGT?GGC?GGC?GGC?GGT?GGT?GGC?GGC?AGC?CTC?TCT?TCG?CTG?GCC?AAT?GCC?GGC?GGT
CTG?CAT?GAC?GAC?GGC?CCG?GGT?CTG?GAT?AAC?GAT?CTC?ATG?AAC?GAG?CCC?ATG?GGT?CTC
GGC?GGT?CTG?GGA?GGA?GGT?GGC?GGC?GGT?GGC?GGC?AAG?AAG?CAC?GAC?CGC?GGT?GGC?GGC
GGT?GGT?TCC?GGT?ACG?CGG?AAA?ATG?AGC?AGC?GGT?GGC?GGC?GGC?GGT?GAT?CAC?GAC?CAC
GGT?CTT?TCC?TCC?AAG?GAA?AAA?TAC?GAG?CAG?CAC?AAG?ATC?ACC?AGC?TAC?CTG?ACG?TCC
AAA?GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA?GGA?GGA?GGC?GGC?GGT?TTG?GAT?CGC?AAC?TCC
GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG?GAG?AGC?GAC?AGC?GAG?GAT?TCT?GTA?ACG?TTC
GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA?AAG?TGC?GGC?TAA
Synthetic be used to the to increase upstream primer of gp52 PROTEIN C end antigen epitope genes is P1 (808-825bp): 5 '-CGGGATCCGAACCTTTCCAGCGTGGC-3 '; Downstream primer is P2 (1285-1302bp): 5 '-CGAATTCTTAGCCGCAGTTTTGCTTCTT-3 '.Synthetic primer deionized water dissolving makes the primer final concentration be 100pmol/ μ l.
Get HCMV (the AD169 strain is provided by Chinese pharmaceutical biological product) cells and supernatant 200 μ l, add 100 μ l Proteinase Ks (10mg/ml), put 56 ℃ of water-baths 4 hours.With phenol/chloroform method purifying HCMV DNA, the HCMV DNA that obtains is dissolved in the 20 μ l deionized waters, put-20 ℃ frozen standby.
The HCMV DNA of above-mentioned purification is a template, gene fragment with primer P1, P2 pcr amplification gp52 PROTEIN C end epitope, reaction density is: HCMV DNA is template 2 μ l, primer P1, P2 each 1 μ l, 10xBuffer 5.0 μ l, 2.5mmol/L dNTP 4.0 μ l, Taq archaeal dna polymerase 0.5 μ l (2.5U), deionized water 36.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations; Last 72 ℃ were extended 7 minutes.Electrophoresis reclaims, the gene fragment of purifying amplification, is dissolved in the 20 μ l deionized waters, put-20 ℃ frozen standby.
3. expressed fusion protein construction of recombinant plasmid:
Extract plasmid pET28a (+) (U.S. Novagen company product),, reclaim the big fragment of plasmid that enzyme is cut behind the electrophoresis, be dissolved in the 20 μ l deionized waters with Nco I and EcoR I double digestion.
The positive and negative chain of gene fragment of synthetic PP150 C end polypeptide is dissolved in respectively in the damping fluid (50mmol/L Tris-HClpH7.4,50mmol/L NaCl), and the final concentration that dissolves back two chains is identical.Get the positive and negative chain solution of equivalent and place mixing in the same centrifuge tube, put 100 ℃ of water-soluble interior sex change 5min, take out rearmounted room temperature naturally cooling, making two chain renaturation is double-stranded gene fragment, and its 5 ' end forms Nco I sticky end, and 3 ' end forms BamH I sticky end.
Take the gp52 gene fragment of EcoR I and BamH I double digestion purifying, electrophoresis is dissolved in the 20 μ l deionized waters after reclaiming.
Get above-mentioned three kinds of enzymes and cut the back dna fragmentation, the volumetric molar concentration mixing such as press, in same centrifuge tube, connect (putting 16 ℃ of connections spends the night) with the T4 dna ligase, making PP150 PROTEIN C end group connect with the gp52 gene fragment to be inserted between carrier pET28a (+) interior the Nco I and EcoR I site because of fragment (makes up flow process and sees Fig. 2), both translate the framework unanimity, express a fusion rotein.
4. the screening of recombinant plasmid:
The recombinant plasmid transformed that the last step was connected arrives e. coli bl21 (DE3), and the converted product coating is contained on the solid LB substratum of kantlex (60 μ g/ml), puts 37 ℃ of overnight incubation.5 transformant bacterium colonies of random choose next day are inoculated into respectively and contain 4ml liquid LB substratum (containing kantlex 60 μ g/ml) and in vitro, put 37 ℃ of shaking culture 6h, get bacterium liquid 1ml, centrifugal receipts bacterium.Use 50 μ l deionized water suspension thalline respectively, boiling water boils 5min, centrifugal (4 ℃, 12000rpm) 5min, get supernatant (in plasmid is arranged) 2 μ l as pcr template, with the normal chain of synthetic PP150 PROTEIN C end polypeptide gene and the downstream primer of amplification gp52 gene fragment is that primer is right, pcr amplification inserts and carries intravital fusion gene fragment, the PCR reaction density is: plasmid template 2 μ l, each 1 μ l of the normal chain of PP150 PROTEIN C end polypeptide gene and primer P2,10xBuffer 5.0 μ l, 2.5mmol/L dNTP 4.0 μ l, Taq archaeal dna polymerase 0.5 μ l (2.5U), deionized water 36.5 μ l, cumulative volume 50 μ l.Amplification condition is: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations; Last 72 ℃ were extended 7 minutes.Get pcr amplification product 5 μ l, the Agarose gel detection with 1.2%, the result, 5 transformants all amplify the target gene fragment (see figure 3) of 591bp, and the contrast bacterium that contains plasmid pET28a (+) does not amplify this gene fragment.Tentative confirmation, these 5 transformants all contain the gp52 gene fragment of placed in-line PP150 PROTEIN C end polypeptide gene and amplification.
5. the Screening and Identification of expressed fusion protein engineering bacteria:
Above-mentioned 5 positive transformants that will contain recombinant plasmid, be seeded to respectively and contain 3ml LB substratum (containing kanamycin 60 μ g/ml) in vitro, 37 ℃ of shaking culture 3h add IPTG to final concentration 0.5mmol/L, continue shaking culture and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects.Electrophoresis detection shows (Fig. 4), and 5 recons are all expressed relative molecular weight and are about 22000 fusion rotein, and expression amount is about 30%～38%, and contrast bacterium BL21 (DE3) does not have this protein band.Obtained the engineering bacteria of 25 amino acid of high expression level human cytomegalic inclusion disease virus PP150 PROTEIN C end and 164 amino acid fusion roteins of gp52 PROTEIN C end.
6.DNA The sequencing results:
To express No. 1 transformant of target protein, extract plasmid and carry out determined dna sequence, the PP150 PROTEIN C end polypeptide gene in the plasmid and the dna sequence dna of gp52 gene fragment are entirely true, and the result is as follows:
ATG?GGC?GGT?GCT?AAA?ACT?CCG?TCT?GAC?GCT?GTT?CAG?AAC?ATC?CTG?CAG?AAA?ATC?GAA
AAA?ATC?AAA?AAC?ACT?GAA?GAA?GGA?TCC?GAA?CCT?TTC?CAG?CGT?GGC?GAT?CCC?TTC?GAC
AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC?AAG?TCG?CGT?GGC?GGC?GGC?GGT?GGT?GGC?GGC?AGC
CTC?TCT?TCG?CTG?GCC?AAT?GCC?GGC?GGT?CTG?CAT?GAC?GAC?GGC?CCG?GGT?CTG?GAT?AAC
GAT?CTC?ATG?AAC?GAG?CCC?ATG?GGT?CTC?GGC?GGT?CTG?GGA?GGA?GGT?GGC?GGC?GGT?GGC
GGC?AAG?AAG?CAC?GAC?CGC?GGT?GGC?GGC?GGT?GGT?TCC?GGT?ACG?CGG?AAA?ATG?AGC?AGC
GGT?GGC?GGC?GGC?GGT?GAT?CAC?GAC?CAC?GGT?CTT?TCC?TCC?AAG?GAA?AAA?TAC?GAG?CAG
CAC?AAG?ATC?ACC?AGC?TAC?CTG?ACG?TCC?AAA?GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA?GGA
GGA?GGC?GGC?GGT?TTG?GAT?CGC?AAC?TCC?GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG?GAG
AGC?GAC?AGC?GAG?GAT?TCT?GTA?ACG?TTC?GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA?AAC
TGC?GGC?TAA
The purifying of express recombinant human cytomegalovirus fusion protein
Material and method
1. main agents: DEAE-SepharoseFF negatively charged ion and S-SepharoseFF positively charged ion gel are Pharmacia company product, and IPTG, DTT are Promega company product.Other reagent is homemade or the import analytical reagent.
2. express the proteic ultrasonic degradation of gp52: the centrifugal (8000rpm of the proteic engineering bacteria of expression gp52 that will cultivate, 10mins, 4 ℃), abandon supernatant, thalline is resuspended in the lysate (50mmol/L Tris-HClpH8.0,10mmol/L EDTA, 10mmol/L DTT) of original fluid 1/10 volume, and ice-bath ultrasonic is broken bacterium 10mins, centrifugal (12000rpm, 20mins, 4 ℃) collect supernatant, abandon precipitation.The supernatant of collecting is used for next step ion-exchange purification.
3.DEAE-Sepharoe FF anion column purifying: with DEAE-Sepharose FF anion column (the high 20cm of glue, diameter 1.2cm) is connected to Bio-Rad normal pressure chromatographic system, earlier with balance liquid (50mmol/LTris-HCl pH8.0,1mmol/L EDTA, 0.1mMmol/L DTT) flushing balance, to go up the cracking supernatant of step acquisition then and directly go up sample, last sample flow velocity is 1.5ml/min.Wash baseline before stylus is back to sample behind the last sample with balance liquid.Successively with contain 50,100,150,200,250,300, the balance liquid eluted protein of 400mmol/L and 1mol/L NaCl, collect each elution peak albumen respectively and carry out SDS-PAGE and detect.
4.S-Sepharose FF cation seperation column purifying: that will go up that step DEAE-Sepharose FF negatively charged ion purifying obtains contains the proteic elution peak of gp52 (50mmol/L NaCl peak), pack in the dialysis tubing, to 20mmol/L PB (pH7.0), 1mmol/L EDTA, 0.1mmol/L DTT solution stirring dialysis 4h.(the high 15cm of glue, diameter 1.2cm) is connected to BioRad normal pressure chromatographic system with the S-SepharoseFF cation seperation column, and with above-mentioned PB solution flushing balance, the gp52 albumen after will dialysing is then directly gone up sample, last sample flow velocity 1.5ml/min.Fully wash post with PB solution behind the end of the sample, make stylus be back to baseline, successively with contain 50,100,200, the PB eluant solution albumen of 300mmol/L and 1mol/L NaCl, collect each elution peak albumen progress SDS-PAGE and detect.
The result
1.DEAE-Sepharose FF anion column purifying gp52 albumen
Each peak albumen of different concns NaCl wash-out carries out SDS-PAGE and analyzes demonstration, and gp52 albumen mainly is present in the 100mmol/L NaCl elution peak, does not have this protein band in other concentration NaCl elution peak.This step obtains the existing very high purity (see figure 5) of gp52 albumen.
2.S-Sepharose FF cation seperation column purified fusion protein
Different concns NaCl albumen of wash-out from the S-Sepharose FF post is carried out SDS-PAGE to be analyzed, the result shows, gp52 albumen exists only in (see figure 5) in the 100mmol/L NaCl elution peak, a dense gp52 protein band (22kD place) develops the color, only a very weak gp52 proteolytic degradation band is slightly before arranged, no gp52 protein band (see figure 5) in other elution peak at it.
The evaluation of purification of Recombinant human cytomegalovirus fusion protein and application
The recombinant human cytomegalovirus fusion protein of purifying is used as antigen, by immunoblotting (Western Blot) and indirect ELISA test method, detect HCMV antibody (IgM or IgG) positive and negative serum, with specificity and the antigenicity of identifying this recombinant protein antigen.Experimental result shows that this recombinant protein has excellent specificity and antigenicity, can be used as Detection of antigen HCMV antibody.
Material and method
1. anti-HCMV IgM positive serum: provide by hospital general microbial room of Nanjing Military Command.
2. integrated enzyme reaction material: elisa plate is that 96 orifice plates are produced in Shenzhen, the mouse-anti people μ chain monoclonal antibody of horseradish peroxidase (HRP) mark is provided by hospital general microbial room of Nanjing Military Command, and the mouse-anti people μ chain monoclonal antibody of alkaline phosphatase (AKP) mark is available from crystalline substance U.S. company.Other material is the conventional material of integrated enzyme reaction.
3.ELISA test: adopt the IgM antibody in the indirect ELISA detection serum.Basic step is: dilute the recombinant protein bag by elisa plate with 50mmol/L carbonate solution (pH9.6), and every hole 100 μ l, 4 ℃ are spent the night.Inferior daily confining liquid (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.5%casein, 0.05% sulphur sulphur mercury, 0.5% sucrose) sealing, every hole 130 μ l, 37 ℃ of 1h (or 4 ℃ spend the night).With anti-HCMVIgM the moon to be measured, positive serum sample diluent (10mmol/L phosphate buffered saline buffer, pH7.4,10% lowlenthal serum, 20% calf serum, 0.5%casein, 0.05% sulphur sulphur mercury, 0.5mmol/L NaCl) after the dilution in 1: 100, add in the enzyme linked plate holes after the sealing every hole 100 μ l respectively, each sample adds 2 holes, 37 ℃ of reaction 30min are with PBST liquid (10mmol/L phosphate buffered saline buffer, pH7.4,0.5% tween 20) wash 5 times after, the anti-people μ chain monoclonal antibody that adds the horseradish peroxidase-labeled of dilution in 1: 5000, every hole 100 μ l, 37 ℃ of reaction 30min, PBST washes 5 times, add substrate TMB solution 100 μ l, 37 ℃ of lucifuge colour developing 10min add 50 μ l 4N sulfuric acid mixing termination reactions, measure the A450 value with enzyme connection instrument.
4. western blot test: recombinant protein of purifying etc. is carried out 12%SDS-PAGE, after electrophoresis finishes, electrotransfer is to nitrocellulose filter, with the PBS liquid that contains 1% bSA (10mmol/L phosphate buffered saline buffer, pH7.4) room temperature vibration envelope film 2h, film is moved in the anti-HCMV-IgM positive or negative serum that dilutes at 1: 50 with PBS liquid 37 ℃ of reaction 1h.Take out film with PBST liquid (10mmol/L phosphate buffered saline buffer, pH7.4,0.5% tween 20) fully after the washing, move in the mouse-anti people μ chain monoclonal antibody solution with the alkali phosphatase enzyme mark of PBS liquid dilution in 1: 1,000 37 ℃ of reaction 1h.Add substrate tolidine (DAB) solution after taking out the film thorough washing, color development at room temperature 5min rinses well with deionized water.
The result
1. indirect ELISA detects the HCMV IgM antibody in the serum
With the PP150 of purifying and 1: 500～1: 8000 doubling dilution bag of gp52 fusion rotein by elisa plate, detect known 1 part of anti-gp52 IgM positive serum, 1 part of anti-PP150 IgM positive serum and 1 portion of normal human serum, the result shows (seeing Table 1), fusion rotein can react with anti-gp52 IgM and anti-PP150 IgM positive serum,, do not illustrate that tentatively this fusion rotein has antigenicity and specificity preferably with the normal human serum reaction.
The ELISA experimental result of table 1 fusion rotein (A450 value)
Bag was by fusion rotein 1: 500 1: 1,000 1: 2,000 1: 4,000 1: 8000
Anti-gp52 IgM positive serum 1.258 0.984 0.815 0.468 0.294
Anti-PP150 IgM positive serum 0.956 0.785 0.688 0.391 0.180
Normal human serum 0.070 0.049 0.042 0.028 0.024
The fusion rotein of purifying and the bovine serum albumin of known different concns (the 5th part) co-electrophoresis, colour developing back are compared, determine that the recombination fusion protein concentration of purifying is about 1.5mg/ml.By elisa plate, detect known 4 parts of anti-HCMV-IgM negative serums and 8 parts of anti-HCMV-IgM positive serums with 1: 1000 dilution back of 50mmol/L carbonate solution (pH9.6) bag.The result shows that 4 parts of anti-HCMV-IgM negative serums all do not develop the color, and color reaction all appears in 8 parts of anti-HCMV-IgM positive serums, illustrates that this fusion rotein has antigenicity and specificity preferably.The A450 value of measuring each hole sample with enzyme connection instrument sees Table 2.Detected 184 portions of normal human serums in addition again, their A450 value has confirmed further that all less than 0.05 this fusion rotein has specificity preferably.
Table 2 purified fusion protein ELISA experimental result (A450) *
1 2 3 4 5 6 7 8 9 10 11 12
0.029?0.033?0.041?0.029?0.389?0.467?0.502?0.593?0.708?0.833?1.182?1.393
0.031?0.028?0.039?0.026?0.373?0.493?0.531?0.584?0.696?0.871?1.237?1.460
1～4: anti--HCMV IgM negative serum, 5～12: anti--HCMV IgM positive serum
2. immunoblotting result
The recombination fusion protein of purifying is carried out 12%SDS-PAGE, add 3 road recombination fusion proteins and carry out electrophoresis, electrotransfer is to the nitrocellulose filter fused antigen then.Transfer there are 3 road recombination fusion protein nitrocellulose filters by 3 of swimming lanes, are respectively applied for the immunoblotting assay of following 3 kinds of serum: anti-HCMV gp52 protein I gM positive serum; Anti-PP150 protein I gM positive serum; The normal human serum reaction.The immunoblotting result shows, this fusion rotein can produce specific reaction with anti-gp52 IgM positive serum and anti-PP150 IgM positive serum, orange precipitation band (seeing Fig. 6, shown in the arrow) appears in the fusion rotein place on nitrocellulose filter, does not react with normal human serum.Confirm that further this fusion rotein has antigenicity and specificity preferably.
The sequence table of recombinant human cytomegalovirus fusion protein
＜110〉Li Yuexi
＜120〉recombinant human cytomegalovirus fusion protein and preparation method thereof, application
<160>2
<210>1
<211>192
<212>PRT
＜213〉artificial sequence
<220>
<221>
<222>
＜223〉a kind of recombinant human cytomegalovirus fusion protein, the fusion rotein that to be human cytomegalic inclusion disease virus PP150 PROTEIN C end form from 433 amino acid whose 164 amino acid series connection of the 270th amino acid to the from 1048 amino acid whose 25 amino acid of the 1024th amino acid to the and gp52 PROTEIN C end, 25 amino acid of PP150 PROTEIN C end are held at fusion rotein N, 164 amino acid of gp52 PROTEIN C end are at the C end of fusion rotein N end fusion rotein, between the two by 2 amino acid of glycine (Gly) and Serine (Ser) (be fusion rotein the 27th with 28 amino acid) be connected, wherein the 162nd amino acid (being the 190th amino acid of fusion rotein) in 164 amino acid of gp52 PROTEIN C end replaces with asparagus fern acyl acid (Asn) by Methionin (Lys), the N end (being the 1st amino acid of fusion rotein) of fusion rotein has increased by 1 egg base acid (Met), 192 amino acid of total length.
<400>1
Met?Gly?Gly?Ala?Lys?Thr?Pro?Ser?Asp?Ala?Val?Gln?Asn?Ile?Leu?Gln
1 5 10 15
Lys?Ile?Glu?Lys?Ile?Lys?Asn?Thr?Glu?Glu?Gly?Ser?Glu?Pro?Phe?Gln
20 25 30
Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser?Gly?Lys?Ser
35 40 45
Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn?Ala
50 55 60
Gly?Gly?Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn
65 70 75 80
Glu?Pro?Met?Gly?Leu?Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly
85 90 95
Lys?Lys?His?Asp?Arg?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met
100 105 110
Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp?His?Asp?His?Gly?Leu?Ser?Ser?Lys
115 120 125
Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu?Thr?Ser?Lys?Gly
130 135 140
Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn
145 150 155 160
Ser?Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp
165 170 175
Ser?Val?Thr?Phe?Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Asn?Cys?Gly
180 185 190
<210>2
<211>579
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)...(579)
<220>
<221>mis-feature
<222>(1)...(3)
The initiator codon that increases when＜223〉synthesizing PP150 PROTEIN C end polypeptide gene.
<220>
<221>mis-feature
<222>(4)...(78)
＜223〉the PP150 PROTEIN C end polypeptide gene sequence of synthetic.
<220>
<221>mis-feature
<222>(79)...(84)
＜223〉the EcoR I restriction enzyme site of connection PP150 PROTEIN C end polypeptide gene and gp52 PROTEIN C end fragment gene.
<220>
<221>mis-feature
<222>(85)...(579)
＜223〉gp52 PROTEIN C end fragment gene order, wherein the 570th bit base is c by the g artificial mutation.
<400>2
atg?ggc?ggt?gct?aaa?act?ccg?tct?gac?gct?gtt?cag?aac?atc?ctg?cag?aaa?atc?gaa?aaa 60
Met?Gly?Gly?Ala?Lys?Thr?Pro?Ser?Asp?Ala?Val?Gln?Asn?Ile?Leu?Gln?Lys?Ile?Glu?Lys
1 5 10 15 20
atc?aaa?aac?act?gaa?gaa?gga?tcc?gaa?cct?ttc?cag?cgt?ggc?gat?ccc?ttc?gac?aaa?aat?120
Ile?Lys?Asn?Thr?Glu?Glu?Gly?Ser?Glu?Pro?Phe?Gln?Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn
25 30 35 40
tac?gtc?ggg?aac?agc?ggc?aag?tcg?cgt?ggc?ggc?ggc?ggt?ggt?ggc?ggc?agc?ctc?tct?tcg?180
Tyr?Val?Gly?Asn?Ser?Gly?Lys?Ser?Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser
45 50 55 60
ctg?gcc?aat?gcc?ggc?ggt?ctg?cat?gac?gac?ggc?ccg?ggt?ctg?gat?aac?gat?ctc?atg?aac?240
Leu?Ala?Asn?Ala?Gly?Gly?Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn
65 70 75 80
gag?ccc?atg?ggt?ctc?ggc?ggt?ctg?gga?gga?ggt?ggc?ggc?ggt?ggc?ggc?aag?aag?cac?gac?300
Glu?Pro?Met?Gly?Leu?Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Lys?Lys?His?Asp
85 90 95 100
cgc?ggt?ggc?ggc?ggt?ggt?tcc?ggt?acg?cgg?aaa?atg?agc?agc?ggt?ggc?ggc?ggc?ggt?gat?360
Arg?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met?Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp
105 110 115 120
cac?gac?cac?ggt?ctt?tcc?tcc?aag?gaa?aaa?tac?gag?cag?cac?aag?atc?acc?agc?tac?ctg?420
His?Asp?His?Gly?Leu?Ser?Ser?Lys?Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu
125 130 135 140
acg?tcc?aaa?ggt?gga?tcg?ggc?ggc?ggc?gga?gga?gga?gga?ggc?ggc?ggt?ttg?gat?cgc?aac?480
Thr?Ser?Lys?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn
145 150 155 160
tcc?ggc?aat?tac?ttc?aac?gac?gcg?aaa?gag?gag?agc?gac?agc?gag?gat?tct?gta?acg?ttc?540
Ser?Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp?Ser?Val?Thr?Phe
165 170 175 180
gag?ttc?gtc?cct?aac?acc?aag?aag?caa?aag?tgc?ggc?tag
Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Asn?Cys?Gly
185 190
atgggcggtg?ctaaaactcc?gtctgacgct?gttcagaaca?tcctgcagaa?aatcgaaaaa 60
atcaaaaaca?ctgaagaagg?atccgaacct?ttccagcgtg?gcgatccctt?cgacaaaaat 120
tacgtcggga?acagcggcaa?gtcgcgtggc?ggcggcggtg?gtggcggcag?cctctcttcg 180
ctggccaatg?ccggcggtct?gcatgacgac?ggcccgggtc?tggataacga?tctcatgaac 240
gagcccatgg?gtctcggcgg?tctgggagga?ggtggcggcg?gtggcggcaa?gaagcacgac 300
cgcggtggcg?gcggtggttc?cggtacgcgg?aaaatgagca?gcggtggcgg?cggcggtgat 360
cacgaccacg?gtctttcctc?caaggaaaaa?tacgagcagc?acaagatcac?cagctacctg 420
acgtccaaag?gtggatcggg?cggcggcgga?ggaggaggag?gcggcggttt?ggatcgcaac 480
tccggcaatt?acttcaacga?cgcgaaagag?gagagcgaca?gcgaggattc?tgtaacgttc 540
gagttcgtcc?ctaacaccaa?gaagcaaaag?tgcggctag
Claims (3)
1. recombinant human cytomegalovirus fusion protein, be 25 amino acid of human cytomegalic inclusion disease virus PP150 PROTEIN C end and 164 fusion roteins that the amino acid series connection forms of gp52 PROTEIN C end, 25 amino acid of PP150 PROTEIN C end are held at fusion rotein N, 164 amino acid of gp52 PROTEIN C end are at the C of fusion rotein end, be connected by 2 amino acid of glycine (Gly) and Serine (Ser) between the two, wherein the 162nd amino acid in 164 amino acid of gp52 PROTEIN C end replaces with asparagus fern acyl acid (Asn) by Methionin (Lys), the N end of fusion rotein has increased by 1 egg base acid (Met), 192 amino acid of total length, it is characterized in that 164 amino acid of this fusion rotein C end are selected from the amino acid of gp52 PROTEIN C end 270-433 position, this fusion rotein aminoacid sequence is:
Met?Gly?Gly?Ala?Lys?Thr?Pro?Ser?Asp?Ala?Val?Gln?Asn?Ile?Leu?Gln
5 10 15
Lys?Ile?Glu?Lys?Ile?Lys?Asn?Thr?Glu?Glu?Gly?Ser?Glu?Pro?Phe?Gln
20 25 30
Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser?Gly?Lys?Ser
35 40 45
Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn?Ala
50 55 60
Gly?Gly?Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn
65 70 75 80
Glu?Pro?Met?Gly?Leu?Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly
85 90 95
Lys?Lys?His?Asp?Arg?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met
100 105 110
Ser?Ser?Gly?Gly?Gly?Gly?Gly?Asp?His?Asp?His?Gly?Leu?Ser?Ser?Lys
115 120 125
Glu?Lys?Tyr?Glu?Gln?His?Lys?Ile?Thr?Ser?Tyr?Leu?Thr?Ser?Lys?Gly
130 135 140
Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Leu?Asp?Arg?Asn
145 150 155 160
Ser?Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu?Ser?Asp?Ser?Glu?Asp
165 170 175
Ser?Val?Thr?Phe?Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln?Asn?Cys?Gly
180 185 190
2. the preparation method of the described recombinant human cytomegalovirus fusion protein of claim 1, this fusion rotein is to utilize the genetic engineering technique preparation, concrete grammar is as follows:
HCMV PP150 PROTEIN C end group synthesizes and renaturation because of segmental:
25 amino acid of the proteic C end of HCMV PPl50 are: Gly Gly Ala Lys Thr Pro Ser AspAla Val Gln Asn Ile Leu Gln Lys Ile Glu Lys Ile Lys Asn Thr Glu Glu;
Two complementary dna fragmentations of chemosynthesis, normal chain is: 5 '-
CATGGGC GGT GCT AAA ACT CCG TCTGAC GCT GTT CAG AAC ATC CTG CAG AAA ATC GAA AAA ATC AAA AAC ACT GAA GAAG-3 '; Minus strand is: 5 '-
GATCCTTC TTC AGT GTT TTT GAT TTT TTC GAT TTT CTG CAG GATGTT CTG AAC AGC GTC AGA CGG AGT TTT AGC ACC GCC-3 ', article two, 5 ' of chain end has all carried out phosphorylation, article two, form above-mentioned 25 amino acid whose gene fragments after chain annealing and the renaturation, and form a NcoI sticky end at 5 ' end, form a BamHI sticky end at 3 ' end;
The screening of HCMV gp52 PROTEIN C end epitope and the clone of gene fragment:
By the proteic whole aminoacid sequences of Computer Analysis HCMV gp52, filter out the strong antigen epi-position in the gp52 albumen, promptly from 433 amino acid of the 270th amino acid to the, its aminoacid sequence and dna sequence dna are as follows:
Epitope aminoacid sequence in the screening gp52:
Glu?Pro?Phe?Gln?Arg?Gly?Asp?Pro?Phe?Asp?Lys?Asn?Tyr?Val?Gly?Asn?Ser
Gly?Lys?Ser?Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Ser?Leu?Ser?Ser?Leu?Ala?Asn
Ala?Gly?Gly?Leu?His?Asp?Asp?Gly?Pro?Gly?Leu?Asp?Asn?Asp?Leu?Met?Asn?Glu
Pro?Met?Gly?Leu?Gly?Gly?Leu?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Lys?Lys?His
Asp?Arg?Gly?Gly?Gly?Gly?Gly?Ser?Gly?Thr?Arg?Lys?Met?Ser?Ser?Gly?Gly?Gly
Gly?Gly?Asp?His?Asp?His?Gly?Leu?Ser?Ser?Lys?Glu?Lys?Tyr?Glu?Gln?His?Lys
Ile?Thr?Ser?Tyr?Leu?Thr?Ser?Lys?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Gly?Gly?Gly
Gly?Gly?Gly?Leu?Asp?Arg?Asn?Ser?Gly?Asn?Tyr?Phe?Asn?Asp?Ala?Lys?Glu?Glu
Ser?Asp?Ser?Glu?Asp?Ser?Val?Thr?Phe?Glu?Phe?Val?Pro?Asn?Thr?Lys?Lys?Gln
Lys?Cys?Gly
The dna sequence dna of the proteantigen epi-position of screening:
GAA?CCT?TTC?CAG?CGT?GGC?GAT?CCC?TTC?GAC?AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC
AAG?TCG?CGT?GGC?GGC?GGC?GGT?GGT?GGC?GGC?AGC?CTC?TCT?TCG?CTG?GCC?AAT?GCC
GGC?GGT?CTG?CAT?GAC?GAC?GGC?CCG?GGT?CTG?GAT?AAC?GAT?CTC?ATG?AAC?GAG?CCC
ATG?GGT?CTC?GGC?GGT?CTG?GGA?GGA?GGT?GGC?GGC?GGT?GGC?GGC?AAG?AAG?CAC?GAC
CGC?GGT?GGC?GGC?GGT?GGT?TCC?GGT?ACG?CGG?AAA?ATG?AGC?AGC?GGT?GGC?GGC?GGC
GGT?GAT?CAC?GAC?CAC?GGT?CTT?TCC?TCC?AAG?GAA?AAA?TAC?GAG?GAG?CAC?AAG?ATC
ACC?AGC?TAC?CTG?ACG?TCC?AAA?GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA?GGA?GGA?GGC
GGC?GGT?TTG?GAT?CGC?AAC?TCC?GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG?GAG?AGC
GAC?AGC?GAG?GAT?TCT?GTA?ACG?TTC?GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA?AAG
TGC?GGC?TAA
Select design PCR primer in the both sides of the dna sequence dna of above-mentioned epitope, the upstream primer of design is P1:5 '-CGGGATCCGAACCTTTCCAGCGTGGC-3 '; Downstream primer is P2:5 '-CGAATTCTTAGCCGCAGTTTTGCTTCTT-3 ', on upstream primer, increased the BamHI restriction enzyme site, EcoRI restriction enzyme site and whole subcipher on downstream primer, have been increased, sequence to downstream primer during synthetic primer has been carried out rite-directed mutagenesis, change bases G into by original base C, this codon amino acids coding becomes Asn by Lys;
Get HCMV AD169 strain cells and supernatant, purifying HCMV DNA; HCMV DNA with purifying is a template, and with the gene fragment of primer P1, P2 pcr amplification gp52 PROTEIN C end epitope, amplification condition is: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations; Electrophoresis reclaims, the gene fragment of purifying amplification; The expressed fusion protein construction of recombinant plasmid:
It is double-stranded gene fragment that the positive and negative chain isoconcentration mixing of the gene fragment of synthetic PP150 C end polypeptide is made two chain renaturation; With NcoI and EcoRI double digestion plasmid pET28a (+), with BamHI and EcoRI double digestion gp52 gene fragment; Above-mentioned three kinds of gene fragments are connected with the T4 dna ligase, PP150 PROTEIN C end group is connected with the gp52 gene fragment because of fragment be inserted between carrier pET28a (+) the interior NcoI and EcoRI site, both translate the framework unanimity, express a fusion rotein;
The screening of recombinant plasmid and evaluation:
With the recombinant plasmid transformed intestinal bacteria, the picking transformant, extract plasmid, cut and dna sequence analysis with the NdeI enzyme, filter out and contain PP150 PROTEIN C end group because of the segmental recombinant plasmid of connecting with the gp52 gene, PP150 PROTEIN C end group is because of 5 ' end at the polyphone gene, and the gp52 gene is at 3 ' end, connected by BamHI restriction enzyme site (underscore part) between the two, sequence is as follows:
ATG?GGC?GGT?GCT?AAA?ACT?CCG?TCT?GAC?GCT?GTT?CAG?AAC?ATC?CTG?CAG?AAA
ATC?GAA?AAA?ATC?AAA?AAC?ACT?GAA?GAA
?GGA?TCC?GAA?CCT?TTC?CAG?CGT?GGC?GAT
CCC?TTC?GAC?AAA?AAT?TAC?GTC?GGG?AAC?AGC?GGC?AAG?TCG?CGT?GGC?GGC?GGC?GGT
GGT?GGC?GGC?AGC?CTC?TCT?TCG?CTG?GCC?AAT?GCC?GGC?GGT?CTG?CAT?GAC?GAC?GGC
CCG?GGT?CTG?GAT?AAC?GAT?CTC?ATG?AAC?GAG?CCC?ATG?GGT?CTC?GGC?GGT?CTG?GGA
GGA?GGT?GGC?GGC?GGT?GGC?GGC?AAG?AAG?CAC?GAC?CGC?GGT?GGC?GGC?GGT?GGT?TCC
GGT?ACG?CGG?AAA?ATG?AGC?AGC?GGT?GGC?GGC?GGC?GGT?GAT?CAC?GAC?CAC?GGT?CTT
TCC?TCC?AAG?GAA?AAA?TAC?GAG?CAG?CAC?AAG?ATC?ACC?AGC?TAC?CTG?ACG?TCC?AAA
GGT?GGA?TCG?GGC?GGC?GGC?GGA?GGA?GGA?GGA?GGC?GGC?GGT?TTG?GAT?CGC?AAC?TCC
GGC?AAT?TAC?TTC?AAC?GAC?GCG?AAA?GAG?GAG?AGC?GAC?AGC?GAG?GAT?TCT?GTA?ACG
TTC?GAG?TTC?GTC?CCT?AAC?ACC?AAG?AAG?CAA?AAC?TGC?GGC?TAA
25 amino acid of expression of recombinant plasmid PP150 PROTEIN C end and 164 amino acid whose fusion roteins of gp52 PROTEIN C end of making up, the i.e. fusion rotein of claim 1 shown in interior;
The Screening and Identification of expressed fusion protein engineering bacteria:
The positive transformant that will contain recombinant plasmid, inoculation contains kantlex 60 μ g/ml LB substratum, 37 ℃ of shaking culture 3h, add IPTG and induce 6h, centrifugal collection thalline carries out SDS-PAGE and detects screening, obtains the engineering bacteria of expressed fusion protein, and the fusion rotein relative molecular weight of expression is about 22000, exist with soluble form, expression amount accounts for 35% of tropina;
The purifying of expressed fusion protein:
With the centrifugal receipts of the engineering bacteria of abduction delivering fusion rotein bacterium, ice-bath ultrasonic is broken bacterium, centrifugal collection supernatant, sample DEAE-Sepharose FF anion column on the supernatant of collecting, with 50,100, the NaCl eluant solution albumen of 1000mmol/L gradient concentration, collect 100mmol/L NaCl elution peak albumen, this peak contains the fusion rotein of expression;
100mmol/L NaCl eluted protein with last step DEAE-Sepharose FF anion column, pack in the dialysis tubing, 20mmol/L PB is dialysed, with S-Sepharose FF cation seperation column on the fusion rotein after the dialysis, with 50,100, the NaCl eluant solution albumen of 1000mmol/L gradient concentration, collect 100mmol/LNaCl elution peak albumen, be the fusion rotein of purifying.
3. claim 1 or 2 described recombinant human cytomegalovirus fusion proteins reach in anti-human cytomegalic inclusion disease virus monoclonal antibody of preparation and the application in resist at detection human cytomegalic inclusion disease virus antibody or antigen more.
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| CN101506222B (en) * | 2006-07-14 | 2013-10-02 | 健泰科生物技术公司 | Refolding of recombinant proteins |
| CN102533795B (en) * | 2012-01-10 | 2013-06-05 | 英诺特（唐山）生物技术有限公司 | Recombinant human cytomegalovirus protein and applications thereof |
| CN105510581A (en) * | 2015-12-11 | 2016-04-20 | 郑州安图生物工程股份有限公司 | Cytomegalovirus IgG antibody affinity detection reagent kit |
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