CN100569942C - A kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application - Google Patents

Abstract

The invention discloses a kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application.This alpha-N-Acetylgalactosaminidase is following (a) or protein (b): (a) protein of being made up of the aminoacid sequence shown in the sequence in the sequence table 3; (b) with the aminoacid sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have an alpha-N-Acetylgalactosaminidase active by (a) deutero-protein.This alpha-N-Acetylgalactosaminidase can be changed into the HRBC blood group O type, be changed into Type B by the AB type by the A type.α NAGA can be used for preparing the test kit that is used for the blood group transformation; α NAGA also can be used for preparing general type red blood cell.

Description

A kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application

Technical field

The present invention relates to a kind of alpha-N-Acetylgalactosaminidase and encoding gene thereof and application.

Background technology

1900, Landsteiner found ABO blood group system first and proposes the homotype blood transfusion, lays a good foundation for modern times blood transfusion medical science, and therefore Landsteiner obtains the nineteen thirty Nobel prize.The antigenic determinant of nineteen sixty Witkins proof ABH is a carbohydrate, and the understanding of people to blood group has been deepened in this discovery.Nearly two during the last ten years, and by using other subject advanced person's analytical procedure, blood transfusion medical science obtains tremendous development, has brought into play huge effect in ensureing people's life safety.Yet transfusion safety and blood bias tyre are still two hang-ups of pendulum in face of us.For a long time, identify that because of blood group the mistake blood transfusion incidence that subjective and objective factors such as mistake, identification error cause remains high, in the flourishing U.S. of science and technology, mistake blood transfusion incidence reaches more than 1 example/1.2 ten thousand units, wherein, the error rate that abo blood group does not conform to is 1 example/3.3 ten thousand units, about 1 example of mortality ratio/800,000 units, be the dead first cause of blood transfusion, more much higher than blood transfusion (as HIV, the HBV etc.) incidence (1 example/2,000,000 units) that spreads disease.Because present erythrocytic preservation period 6 weeks only, blood sampling volume significantly reduces when running into situation such as meropodium holiday, winter and summer vacation, is easy to occur that certain blood group RBC supply is in an emergency and other type RBC is superfluous even expired so that the bias tyre phenomenon of waste.The blood bias tyre not only jeopardizes the life security that needs the blood transfusion patient, also causes huge waste, and U.S.'s statistics reaches 6% more than, and this was to making the matter worse beyond doubt with regard to not abundant blood source originally.

Human erythrocyte's blood group system has 29 kinds at least, and is the most important to transfusion safety with ABO and Rh blood group system.O type RBC does not contain A or B antigen, under the prerequisite of RhD blood group coupling, can be defeated by the blood recipient of A, B, AB or O type safely, and " universal RBC " therefore is otherwise known as.Use universal RBC and can reduce greatly, improve the security of blood transfusion because of the transfusion reaction of abo blood group due to not conforming to; Solve a blood bias tyre difficult problem, reduce the blood waste; Simplify blood collection, store and join the type program, improve the working efficiency of blood station and hospital's Blood Transfusion Department, it is nearly 9% that the blood transfusion cost can be saved, and it is reported that the U.S. can save 700,000,000 dollars every year, and conservative estimation China can save 300,000,000 yuan every year.The Blood Center science person in charge Garratty of American Red Cross estimates to be expected to A, B or AB type RBC are transformed into the O type in the coming years, will only store when the time comes and use O type RBC, and this will be a revolution of blood transfusion medical science.In addition, in war, the attack of terrorism, huge disaster and other accident, the blood transfusion of safety in time is the treatment wounded's a key factor.When using universal RBC, need not to check blood recipient's abo blood group, easy to use, rapid, safety can strengthen emergent blood transfusion supportability, improve the wounded's survival rate.

The ABH blood group antigen be in the nature glycoprotein and glycolipid, blood group is by the kind of its terminal sugar chain and the decision that puts in order.O type RBC only expresses H antigen; Type B RBC only contains B antigen, and B antigen non-reducing end is Duoed α-1,3 semi-lactosi than H antigen; A type RBC is comparatively complicated, mainly is divided into two kinds of hypotypes of A1 and A2.On the A1 blood subgroup red corpuscle, contain A and A1 antigen, and only contain A antigen on the A2 type red corpuscle.The A antigen on A2 blood subgroup RBC surface is Duoed a α-1 than H antigen, the 3-N-acetylgalactosamine, and the A1 antigen on A1 blood subgroup RBC surface is Duoed a N-acetylgalactosamine α 1 → 3 (Fucose α 1 → 2) semi-lactosi [GalNAc α 1 → 3 (Fuc α 1 → 2) Gal] trisaccharide structure than A antigen, and China's A1 blood subgroup accounts for the A type more than 99%; AB type RBC then contains two kinds of antigens of A, B.

The glycoside hydrolase enzymolysis process is the most promising method that realizes general type red blood cell at present.Glycoside hydrolase exists extensively, and kind is abundant.The substrate classification is cut according to mechanism of action and enzyme by international biological chemistry and molecular biology federation, definite designation 150 multiclass glycoside hydrolases.These glycoside hydrolase parts are from higher animal, yeast, aspergillus niger etc., these enzymes all are lysosomal enzymes, optimal pH is between 3.5～4.5, specific enzyme activity generally is no more than 50U/mg, the zymoprotein of reaction needed high density (＞3mg/mL) and low pH value, enzymolysis is incomplete, can not satisfy blood group and change requirement.The part glycoside hydrolase derives from the microorganisms such as clostridium perfringens, Salmonellas, streptococcus pneumoniae, bifidus bacillus, campylobacter jejuni, Flavobacterium, fecal bacteria.Alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase) can respectively the N-acetylgalactosaminase of A antigen, A1 antigen non-reducing end be cut, the H antigen of generation O type erythrocyte surface.

Summary of the invention

The purpose of this invention is to provide a kind of new alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase).

Alpha-N-Acetylgalactosaminidase provided by the present invention (α-N-acetylgalactosaminidase), name is called α NAGA, deriving from the golden yellow bacillus of meningeal sepsis (Chryseobacterium meningosepticum) ZYP01CGMCC No.2198, is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 3;

(b) with the aminoacid sequence of sequence in the sequence table 3 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase) active by (a) deutero-protein.

The golden yellow bacillus of meningeal sepsis (Chryseobacterium meningosepticum) ZYP01CGMCCNo.2198 separated from Chinese clinical samples and obtains, and had been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at Da Tun road, Chaoyang District, BeiJing, China city on October 17th, 2007.

Wherein, the sequence in the sequence table 3 is made up of 427 amino-acid residues.

In order to make the α NAGA in (a) be convenient to purifying, proteinic N end or C end that can the aminoacid sequence shown in the sequence 3 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in α NAGA synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of α NAGA in above-mentioned (b) can pass through SEQ ID № in the sequence table: 1 the codon that lacks one or several amino-acid residue in the dna sequence dna shown in 5 ' terminal the 52nd to 1332 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

For the ease of proteic soluble-expression, signal peptide sequence on also can adding at the N-terminal of described α NAGA.As adding, obtain the albumen of forming by the aminoacid sequence shown in 2 in the sequence table by 2 the polypeptide of forming from N-terminal the 1st to 17 amino acids residue in the sequence table.

Sequence 2 in the sequence table is made up of 444 amino-acid residues.From N-terminal (N end) 1-17 amino acids residue is signal peptide (signal peptide) sequence.

(encoding gene of α-N-acetylgalactosaminidase) also belongs to protection scope of the present invention to above-mentioned alpha-N-Acetylgalactosaminidase.

Described alpha-N-Acetylgalactosaminidase (encoding gene of α-N-acetylgalactosaminidase) specifically can be following 1) or 2) or 3) gene:

1) its encoding sequence be in the sequence table sequence 1 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 52-1332 position;

2) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the described alpha-N-Acetylgalactosaminidase of the encoding (dna molecular of α-N-acetylgalactosaminidase).

Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, at 65 ℃ down with NC film or pvdf membrane hybridization and wash.

Sequence 1 in the sequence table is made up of 1335 deoxyribonucleotides, from 5 ' the 52nd to 1332 the terminal encoding sequence that Nucleotide is the α NAGA of sequence 3, is the encoding sequence of signal peptide from 5 ' terminal 1-51 position Nucleotide.

The recombinant expression vector, transgenic cell line and the reorganization bacterium that contain gene of the present invention all belong to protection scope of the present invention.

Described recombinant expression vector specifically can be with nucleotide sequence be sequence 1 insert the recombinant expression vector pET-22b-α NAGA that the multiple clone site of pET-22b (+) obtains from 5 ' the 52nd to 1332 terminal Nucleotide.

Another object of the present invention provides the above-mentioned alpha-N-Acetylgalactosaminidase of a kind of expression (method of α-N-acetylgalactosaminidase).

The above-mentioned alpha-N-Acetylgalactosaminidase of the expression provided by the present invention (method of α-N-acetylgalactosaminidase), be to contain above-mentioned alpha-N-Acetylgalactosaminidase (recombinant expression vector of the encoding gene of α-N-acetylgalactosaminidase) imports host cell, expresses to obtain alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase).

Described host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.

Described intestinal bacteria specifically can be BL21 (DE3), E.coli JM109, E.coli HB101 or E.coliTop10 etc.

When described host was intestinal bacteria, the carrier that sets out that is used to make up described recombinant expression vector can be existing at above-mentioned expression in escherichia coli expression of exogenous gene carrier.

Above-mentioned recombinant expression vector all can make up according to ordinary method.

Cultivation contains alpha-N-Acetylgalactosaminidase of the present invention, and (α-N-acetylgalactosaminidase) substratum and the culture condition of the host cell of encoding gene all can be substratum and the culture condition of cultivating the host that sets out.

Experimental results show that, (the α NAGA of α-N-acetylgalactosaminidase) can A antigen and A1 antigen efficient under neutral pH, enzymolysis people RBC surface fully for this alpha-N-Acetylgalactosaminidase, make it to change into H antigen, thereby realize the transformation of A type RBC to O type RBC.Illustrate that α NAGA can be changed into the HRBC blood group O type, be changed into Type B by the AB type by the A type.α NAGA can be used for preparing the test kit that is used for the blood group transformation; α NAGA also can be used for preparing general type red blood cell.α NAGA has important clinical meaning and vast market prospect, also can become a toolenzyme of glycobiology research.

Description of drawings

Fig. 1 is the PCR product electrophoretogram of the full-length gene of α NAGA and gene fragment (sequence 1 from 5 ' terminal 52-1332 position deoxyribonucleotide)

M:500bp DNA Marker, 1: the full-length gene of α NAGA, 2: α NAGA gene fragment (sequence 1 from 5 ' terminal 52-1332 position deoxyribonucleotide)

Fig. 2 is that the PCR of pMD-18T-α NAGA and pET-22b-α NAGA identifies collection of illustrative plates.

M:500bp DNA Marker; 1: the PCR result of plasmid pMD-18T-α NAGA; 2: the PCR result of plasmid pET-22b-α NAGA.

Fig. 3 is the SDS-PAGE electrophorogram

M: protein molecular weight standard; The total protein of 1:pET-22b-α NAGA/ e. coli bl21 (DE3) after 0.4mM IPTG induces; 2:pET-22b-α NAGA/ e. coli bl21 (DE3) does not add IPTG inductive total protein; White protein is through Ni on the bacterium of 3:pET-22b-α NAGA/ e. coli bl21 (DE3) after 0.4mM IPTG induces ²⁺Huge legendary turtle is closed the albumen of affinity chromatography column purification gained; 4: with Ni ²⁺The albumen that huge legendary turtle is closed the affinity chromatography column purification is used the albumen of anion-exchange chromatography post Hitrap Q FF purifying gained again.

Fig. 4 A is for detecting N-acetylgalactosaminase (α-N-acetylgalactosaminidase) to the erythrocytic enzymolysis of A1 blood subgroup with blood grouping reagent

Fig. 4 B is for detecting alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase) to the erythrocytic enzymolysis of A2 blood subgroup with blood grouping reagent

Fig. 5 is a microscopic examination erythrocyte blood type photo

Before the A:A1 hypotype red corpuscle enzymolysis; Behind the B:A1 hypotype red corpuscle enzymolysis 1h; Before the C:A2 hypotype red corpuscle enzymolysis; Behind the D:A2 hypotype red corpuscle enzymolysis 1h.

Fig. 6 detects the erythrocytic blood group of enzymolysis with flow cytometer to detect

Before the A:A1 hypotype red corpuscle enzymolysis; Behind the B:A1 hypotype red corpuscle enzymolysis 1h; Before the C:A2 hypotype red corpuscle enzymolysis; Behind the D:A2 hypotype red corpuscle enzymolysis 1h, E:O type red corpuscle.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions is compiled the condition described in " molecular cloning experiment guide " according to people such as normal condition such as Sambrook usually in the following example, or the condition of advising according to manufacturer.

The clone of embodiment 1, alpha-N-Acetylgalactosaminidase α NAGA encoding gene

With the golden yellow bacillus of meningeal sepsis (Chryseobac terium meningosep ticum) ZYP01 CGMCCNo.2198 inoculation to substratum (the 5g/L yeast extract, the 5g/L peptone, 5g/L NaCl, 3g/LK2HP04, all the other are water.PH7.2) in, 200rpm, 30 ℃ of overnight incubation.Extract genomic dna according to Promega genome DNA extracting reagent kit specification sheets.With the genomic dna that extracts is template, utilize upstream primer (sequence is: 5 '-ATGGGCGCCTTAATTCCC-3 ') and downstream primer (sequence is: 5 '-TTAGTAGTCGTCATTTATTGC-3 ') carry out PCR.The PCR condition is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of sex change 30s then, 50 ℃ of annealing 30s, 72 ℃ are extended 90s, totally 30 circulations; Last 72 ℃ are extended 10min.The PCR product is carried out agarose gel electrophoresis detect, obtain the band (among Fig. 1, swimming lane 1) that molecular weight is about 1335bp.Reclaim test kit with sepharose and reclaim this fragment.Should reclaim fragment and be connected, will connect product transformed into escherichia coli DH5 α competent cell,, obtain containing the segmental recombinant plasmid of recovery according to the carboxylic Bian penicillin resistance label screening positive colony on the pMD18-T carrier with pMD18-T plasmid (Takara company).With T7 on this recombinant plasmid vector and T7 terminator sequence is that primer carries out nucleotide sequencing to it, sequencing result shows that the gene that increases forms (sequence 1 in the sequence table) by 1335 deoxyribonucleotides, its open reading frame (ORF) for sequence 1 in the sequence table from 5 ' terminal the 1st to 1335 deoxyribonucleotide, encoding amino acid sequence is the protein of sequence 2 in the sequence table, and 5 ' of sequence 1 terminal 1-51 position Nucleotide is the encoding sequence of signal peptide in sequence table.In the sequence table sequence 1 from 5 ' terminal the 52nd to 1335 deoxyribonucleotide, encoding amino acid sequence is the protein of sequence 3 in the sequence table.

The recombinant vectors called after pTE-α NAGA from the α NAGA gene of 5 ' terminal 1-1335 position deoxyribonucleotide that will contain sequence 1 in the ordered list.

The expression of embodiment 2, α NAGA

1, the structure that contains the expression vector pET-22b-α NAGA of α NAGA encoding gene

With pTE-α NAGA is template, with upstream primer (sequence is: 5 '-ATCATATGCCAAAAAAAGTAAGAATTGC-3 ' (having Nde I restriction enzyme site)), and downstream primer (sequence is: 5 '-TGCTCGAGGTAGTCGTCATTTATTGC-3 ' (having Xho I restriction enzyme site) pcr amplification nucleotide sequence is the α NAGA gene fragment from 5 ' terminal 52-1332 position deoxyribonucleotide of sequence 1 in the sequence table.The PCR condition is: 95 ℃ of pre-sex change 5min of elder generation; 95 ℃ of sex change 30s then, 43 ℃ of annealing 30s, 72 ℃ are extended 90s, totally 30 circulations; Last 72 ℃ are extended 10min.The PCR product is carried out agarose gel electrophoresis detect, obtain the band (among Fig. 1, swimming lane 2) that molecular weight is about 1300bp.

According to the operational guidance of TAKARA company, the PCR product is connected transformed into escherichia coli DH5 α with the pMD-18T carrier.Select positive colony, extract plasmid, identify the plasmid that makes up with enzyme cutting method, PCR method and order-checking, will contain nucleotide sequence and be the recombinant vectors called after pMD-18T-α NAGA from the α NAGA gene fragment of 5 ' terminal 52-1332 position deoxyribonucleotide of sequence 1 in the sequence table.Wherein, the PCR qualification result of pMD-18T-α NAGA obtains being about the band of 1300bp shown in swimming lane among Fig. 21.

According to the operational guidance of TAKARA company,, obtain the fragment of about 1300bp with Nde I and Xho I double digestion plasmid pMD-18T-α NAGA; With Nde I and Xho I double digestion plasmid pET-22b (+) (Novagen company), obtain the long linear plasmid of 5364bp that is, downcut the agarose gel that comprises 1300bp and 5364bp DNA respectively, reclaim DNA, connect products therefrom, transformed into escherichia coli DH5 α, select positive colony, extract plasmid, identify the plasmid that makes up with enzyme cutting method, PCR method and order-checking, will contain nucleotide sequence and be the recombinant vectors called after pET-22b-α NAGA from the α NAGA gene fragment of 5 ' terminal 52-1332 position deoxyribonucleotide of sequence 1 in the sequence table.Wherein, the PCR qualification result of pET-22b-α NAGA obtains being about the band of 1300bp shown in swimming lane among Fig. 22.

2, the expression of α NAGA and purifying

With plasmid pET-22b-α NAGA transformed into escherichia coli BL21 (DE3), identify by enzyme cutting method, PCR method and order-checking, screening obtains changing over to the positive cell of pET-22b-α NAGA, and the positive cell of gained is the genetically engineered host cell that can express target protein.This positive bacteria is called pET-22b-α NAGA/ e. coli bl21 (DE3).

With pET-22b-α NAGA/ e. coli bl21 (DE3) streak inoculation in LB solid medium (containing penbritin 100ug/ml) overnight incubation, the single colony inoculation of picking is in 3ml LB liquid nutrient medium (containing penbritin 100ug/ml) overnight incubation, dilute incubated overnight bacterium liquid in LB liquid nutrient medium (containing penbritin 100ug/ml) with 1: 100 volume ratio, be cultured to bacterium liquid OD600 in 37 ℃ 250 rev/mins and reach at 0.8 o'clock, add isopropylthiogalactoside (IPTG) to final concentration 0.4mmol/L, in 30 ℃ of abduction delivering 12h.4 ℃ of centrifugal collection thalline.

Every gram wet thallus adding 10ml binding buffer liquid (the 20mM sodium phosphate buffer, 500mM NaCl, the 20mM imidazoles, pH 7.4), add the 0.2mg/ml N,O-Diacetylmuramidase, 20ug/ml DNAse, 1mM MgCl ₂, 1mM PMSF stirs 30min under the room temperature, multigelation 5 times.The centrifugal 20min of 10000rpm gets supernatant liquor.With sample on the supernatant liquor to equilibrated Ni ²⁺Huge legendary turtle is closed affinity chromatographic column (Histrap FF crude chromatography column, 1mL, Pharmacia company, 11-0004-58), with the binding buffer liquid washing of 10 times of column volumes, with elution buffer (20mM sodium phosphate buffer, 0.5M NaCl, 500mM imidazoles, pH7.4) wash-out target protein.With sample Zhiyin ion exchange column Hitrap Q FF on the eluted protein (Hitrap Q FF chromatography column, 5mL, Pharmacia company, 17-5156-01) on, lavation buffer solution is a 20mM Tris damping fluid, pH 8.2; Elution buffer is a 20mM Tris damping fluid, 1M NaCl, and pH 8.2.Eluted product is carried out the SDS-PAGE electrophoresis, and electrophoresis result shows the α NAGA that obtains 50kDa as shown in Figure 3.Target protein under wash-out enzymolysis damping fluid (200mM glycine, 3mMNaCl, pH6.8) with ultrafiltration pipe ultrafiltration (Amicon Ultra-15 centrifuge tube, 15ml, molecular weight cut-off 50kd, article No.: UFC900508, Millipore company), detecting enzyme solution purity with HPLC is 98%, uses BCA protein quantification test kit (pierce company, article No. 23225) to determine that concentration is 800ug/ml.

Embodiment 2, change HRBC into the O type by the A type with alpha-N-Acetylgalactosaminidase

Compress red corpuscle 1 time with physiological saline washing 0.5ml people A1 blood subgroup compression red corpuscle and 0.5ml people's A2 blood subgroup respectively, use enzymolysis damping fluid (glycine 200mM respectively, NaCl 3mM, pH6.8) washing people's A1 blood subgroup red corpuscle and people's A2 blood subgroup red corpuscle are 2 times, get the 1ml hematocrit respectively and be 20% A1 blood subgroup red corpuscle and A2 blood subgroup red corpuscle, (200ug of α-N-acetylgalactosaminidase), room temperature was placed 1 hour, rocked therebetween twice to add alpha-N-Acetylgalactosaminidase.Detect erythrocyte blood type with anti-A blood grouping reagent (Changchun Boulder biotech company, catalog number (Cat.No.) s10960081, lot number 20060809), anti-A1 monoclonal antibody (Canadian Dominion Biology company, cat. no AL18001A) and flow cytometer.

1, detects erythrocyte blood type with blood grouping reagent

Get before the enzymolysis or the red corpuscle 40ul behind the enzymolysis, add the anti-A of equivalent (40ul), anti-A1, anti-B and anti-H antibody respectively, abundant mixing, observations behind the 5min.Get the 10ul microscopic examination.The blood grouping reagent detected result is shown in Fig. 4 A and Fig. 4 B, and (A1 red corpuscle and anti-A, the strong aggegation of anti-A1 before the enzymolysis of α-N-acetylgalactosaminidase) are with aggegation a little less than the anti-H, with not aggegation of anti-B for alpha-N-Acetylgalactosaminidase.A1 blood subgroup red corpuscle behind the enzymolysis and anti-A, anti-A1, not aggegation of anti-B are with the strong aggegation of anti-H; A2 red corpuscle and the strong aggegation of anti-A before the enzymolysis are with aggegation a little less than the anti-H, with anti-A1, not aggegation of anti-B.A2 blood subgroup red corpuscle behind the enzymolysis and anti-A, anti-A1, not aggegation of anti-B are with the strong aggegation of anti-H.

The microscopic examination result shows that the preceding A1 of enzymolysis, A2 blood subgroup red corpuscle and anti-A antibody form big grumeleuse, A1, A2 blood subgroup and all not aggegations of anti-A antibody behind the enzymolysis as shown in Figure 5.

Illustrate α NAGA with the A antigen on A1 blood subgroup and A2 blood subgroup RBC surface and

Outermost GalNAc α 1, the 3 glycosidic link fracture of the A1 antigen on A1 blood subgroup RBC surface decomposes α-N-acetylgalactosamine, makes A1 and A antigen all be converted into H antigen.Illustrate that α NAGA is a kind of alpha-N-Acetylgalactosaminidase (α-N-acetylgalactosaminidase).

2, detect erythrocyte blood type with flow cytometer

Get before the enzymolysis or the red corpuscle behind the enzymolysis, with physiological saline washing 2 times, with PBS washing 1 time, add the anti-A serum of 100ul people (tire is 1: 64), 37 ℃ of water-bath 1h, PBS washing 3 times, the anti-human IgG two anti-100ul of FITC labelled goat that add dilution in 1: 100, incubated at room 1h, PBS washing 3 times detects with flow cytometer.Each 10000 cells of sorting that detect.The result shows that the peak behind A1 and the A2 blood subgroup red corpuscle enzymolysis obviously moves to left as shown in Figure 6, and fluorescence significantly weakens, and is consistent with the erythrocytic streaming performance of O type.Illustrate that α NAGA changes red corpuscle into the O type by A1 blood subgroup and A2 blood subgroup respectively.

Sequence table

<160>3

<210>1

<211>1335

<212>DNA

＜213〉the golden yellow bacillus of meningeal sepsis (Chryseobacterium meningosepticum)

<400>1

atgggcgcct taattccctc gagctcatta ttcaacattt tcgattttaa tccaaaaaaa 60

gtaagaattg cttttatagc agttggttta cgcggacaga ctcacgtaga aaacatggcg 120

agacgcgatg acgtagaggt tgtggctttt gcagatccgg atccgtacat ggtaggccgt 180

gcgcaggaaa tcctgaaaaa gaatggcaga aaacctgcta aagtttttgg aaacggaaat 240

tacgactaca aaaacatgct taaagataaa aatatcgatg ctgtttttgt atcctctcca 300

tgggaatggc accatgaaca tggtgtagca gccatgaaag ctggtaaaat tgtcggaatg 360

gaggtttgtg gtgctttaac aatggatgag tgctgggatt atgtaaaagt ttccgagcag 420

acaggagttc cgttaatggc tttggaaaat gtatgttaca gacgcgatat tatggctgtc 480

cttaacatgg taagaaaaga catgttcgga gaattaattc gcggaacggg aggctaccag 540

cacgatctca gaccagttct tttcaatagc ggaatcaacg gtaaaaacgg agatggtgtt 600

gagtttggtg acaaagcctt cagtgaagcg aaatggagaa ccaaccatta taaaaacaga 660

aacggggaac tctaccctac tcacggttta ggcccattac acacaatgat ggatattaac 720

cgcgggaaca gattattaag attatcatct tttgcttcca aagcaagagg tttacataaa 780

tatgtcgtgg ataagggcgg ggaaagccat ccaaatgcaa aagtagaatg gaaacaagga 840

gatattgtaa ccactcagat ccagtgtaac aacggagaga ctattgtatt aacccacgat 900

accagcctgc aaagaccata taacctggga ttcaaagttc agggtactga aggtctttgg 960

gaagattttg gctggggaga tgcagcacaa ggatttattt acttcgagaa aataatgaat 1020

cattctcaca gatgggatgg ttcagaaaaa tggatgaaag aatatgacca cccgatgtgg 1080

aaaaaacacg aacagaaagc tgttggtgca ggtcatggcg gaatggatta cttccttgat 1140

aatactttta tcgagtgtat caaacgaaat gaagcattcc cgctggatgt ttacgatctg 1200

gcgacgtggt attccattac acctcttagt gaaaagtcta ttgctgaaaa cggtgccgtt 1260

caggaaattc ctgattctac aaacggtaaa tggaaaactg ctaaaaatac atttgcaata 1320

aatgacgact actaa 1335

<210>2

<211>444

<212>PRT

＜213〉the golden yellow bacillus of meningeal sepsis (Chryseobacterium meningosepticum)

<400>2

Met Gly Ala Leu Ile Pro Ser Ser Ser Leu Phe Asn Ile Phe Asp Phe

1 5 10 15

Asn Pro Lys Lys Val Arg Ile Ala Phe Ile Ala Val Gly Leu Arg Gly

20 25 30

Gln Thr His Val Glu Asn Met Ala Arg Arg Asp Asp Val Glu Val Val

35 40 45

Ala Phe Ala Asp Pro Asp Pro Tyr Met Val Gly Arg Ala Gln Glu Ile

50 55 60

Leu Lys Lys Asn Gly Arg Lys Pro Ala Lys Val Phe Gly Asn Gly Asn

65 70 75 80

Tyr Asp Tyr Lys Asn Met Leu Lys Asp Lys Asn Ile Asp Ala Val Phe

85 90 95

Val Ser Ser Pro Trp Glu Trp His His Glu His Gly Val Ala Ala Met

100 105 110

Lys Ala Gly Lys Ile Val Gly Met Glu Val Cys Gly Ala Leu Thr Met

115 120 125

Asp Glu Cys Trp Asp Tyr Val Lys Val Ser Glu Gln Thr Gly Val Pro

130 135 140

Leu Met Ala Leu Glu Asn Val Cys Tyr Arg Arg Asp Ile Met Ala Val

145 150 155 160

Leu Asn Met Val Arg Lys Asp Met Phe Gly Glu Leu Ile Arg Gly Thr

165 170 175

Gly Gly Tyr Gln His Asp Leu Arg Pro Val Leu Phe Asn Ser Gly Ile

180 185 190

Asn Gly Lys Asn Gly Asp Gly Val Glu Phe Gly Asp Lys Ala Phe Ser

195 200 205

Glu Ala Lys Trp Arg Thr Asn His Tyr Lys Asn Arg Asn Gly Glu Leu

210 215 220

Tyr Pro Thr His Gly Leu Gly Pro Leu His Thr Met Met AspIle Asn

225 230 235 240

Arg Gly Asn Arg Leu Leu Arg Leu Ser Ser Phe Ala Ser Lys Ala Arg

245 250 255

Gly Leu His Lys Tyr Val Val Asp Lys Gly Gly Glu Ser His Pro Asn

260 265 270

Ala Lys Val Glu Trp Lys Gln Gly Asp Ile Val Thr Thr Gln Ile Gln

275 280 285

Cys Asn Asn Gly Glu Thr Ile Val Leu Thr His Asp Thr Ser Leu Gln

290 295 300

Arg Pro Tyr Asn Leu Gly Phe Lys Val Gln Gly Thr Glu Gly Leu Trp

305 310 315 320

Glu Asp Phe Gly Trp Gly Asp Ala Ala Gln Gly Phe Ile Tyr Phe Glu

325 330 335

Lys Ile Met Asn His Ser His Arg Trp Asp Gly Ser Glu Lys Trp Met

340 345 350

Lys Glu Tyr Asp His Pro Met Trp Lys Lys His Glu Gln Lys Ala Val

355 360 365

Gly Ala Gly His Gly Gly Met Asp Tyr Phe Leu Asp Asn Thr Phe Ile

370 375 380

Glu Cys Ile Lys Arg Asn Glu Ala Phe Pro Leu Asp Val Tyr Asp Leu

385 390 395 400

Ala Thr Trp Tyr Ser Ile Thr Pro Leu Ser Glu Lys Ser Ile Ala Glu

405 410 415

Asn Gly Ala Val Gln Glu Ile Pro Asp Ser Thr Asn Gly Lys Trp Lys

420 425 430

Thr Ala Lys Asn Thr Phe Ala Ile Asn Asp Asp Tyr

435 440

<210>3

<211>427

<212>PRT

＜213〉the golden yellow bacillus of meningeal sepsis (Chryseobacterium meningosepticum)

<400>3

Pro Lys Lys Val Arg Ile Ala Phe Ile Ala Val Gly Leu Arg Gly Gln

1 5 10 15

Thr His Val Glu Asn Met Ala Arg Arg Asp Asp Val Glu Val Val Ala

20 25 30

Phe Ala Asp Pro Asp Pro Tyr Met Val Gly Arg Ala Gln Glu Ile Leu

35 40 45

Lys Lys Asn Gly Arg Lys Pro Ala Lys Val Phe Gly Asn Gly Asn Tyr

50 55 60

Asp Tyr Lys Asn Met Leu Lys Asp Lys Asn Ile Asp Ala Val Phe Val

65 70 75 80

Ser Ser Pro Trp Glu Trp His His Glu His Gly Val Ala Ala Met Lys

85 90 95

Ala Gly Lys Ile Val Gly Met Glu Val Cys Gly Ala Leu Thr Met Asp

100 105 110

Glu Cys Trp Asp Tyr Val Lys Val Ser Glu Gln Thr Gly Val Pro Leu

115 120 125

Met Ala Leu Glu Asn Val Cys Tyr Arg Arg Asp Ile Met Ala Val Leu

130 135 140

Asn Met Val Arg Lys Asp Met Phe Gly Glu Leu Ile Arg Gly Thr Gly

145 150 155 160

Gly Tyr Gln His Asp Leu Arg Pro Val Leu Phe Asn Ser Gly Ile Asn

165 170 175

Gly Lys Asn Gly Asp Gly Val Glu Phe Gly Asp Lys Ala Phe Ser Glu

180 185 190

Ala Lys Trp Arg Thr Asn His Tyr Lys Asn Arg Asn Gly Glu Leu Tyr

195 200 205

Pro Thr His Gly Leu Gly Pro Leu His Thr Met Met Asp Ile Asn Arg

210 215 220

Gly Asn Arg Leu Leu Arg Leu Ser Ser Phe Ala Ser Lys Ala Arg Gly

225 230 235 240

Leu His Lys Tyr Val Val Asp Lys Gly Gly Glu Ser His Pro Asn Ala

245 250 255

Lys Val Glu Trp Lys Gln Gly Asp Ile Val Thr Thr Gln Ile Gln Cys

260 265 270

Asn Asn Gly Glu Thr Ile Val Leu Thr His Asp Thr Ser Leu Gln Arg

275 280 285

Pro Tyr Asn Leu Gly Phe Lys Val Gln Gly Thr Glu Gly Leu Trp Glu

290 295 300

Asp Phe Gly Trp Gly Asp Ala Ala Gln Gly Phe Ile Tyr Phe Glu Lys

305 310 315 320

Ile Met Asn His Ser His Arg Trp Asp Gly Ser Glu Lys Trp Met Lys

325 330 335

Glu Tyr Asp His Pro Met Trp Lys Lys His Glu Gln Lys Ala Val Gly

340 345 350

Ala Gly His Gly Gly Met Asp Tyr Phe Leu Asp Asn Thr Phe Ile Glu

355 360 365

Cys Ile Lys Arg Asn Glu Ala Phe Pro Leu Asp Val Tyr Asp Leu Ala

370 375 380

Thr Trp Tyr Ser Ile Thr Pro Leu Ser Glu Lys Ser Ile Ala Glu Asn

385 390 395 400

Gly Ala Val Gln Glu Ile Pro Asp Ser Thr Asn Gly Lys Trp Lys Thr

405 410 415

Ala Lys Asn Thr Phe Ala Ile Asn Asp Asp Tyr

420 425

Claims (11)

1, a kind of albumen, its aminoacid sequence is shown in sequence in the sequence table 3.

2, the described proteic encoding gene of claim 1.

3, gene according to claim 2 is characterized in that: the described proteic encoding gene of claim 1 is following 1) or 2) dna molecular:

1) in the sequence table sequence 1 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 52-1332 position;

2) dna molecular shown in the sequence 1 in the sequence table.

4, the recombinant expression vector that contains claim 2 or 3 described genes.

5, the transgenic cell line that contains claim 2 or 3 described genes.

6, contain claim 2 or 3 described gene recombination bacteriums.

7, the described proteic method of a kind of expression claim 1 is that the recombinant expression vector that will contain the described protein coding gene of claim 1 imports host cell, expresses obtaining the described albumen of claim 1.

8, method according to claim 7 is characterized in that: described recombinant expression vector for nucleotide sequence be sequence 1 insert the recombinant expression vector pET-22b-α NAGA that the multiple clone site of pET-22b (+) obtains from 5 ' the 52nd to 1332 terminal Nucleotide.

9, method according to claim 8 is characterized in that: described host cell is e. coli bl21 (DE3) cell.

10, the application of the described albumen of claim 1 in preparing the medicine that the HRBC blood group is changed into the O type by the A type.

11, a kind of described proteic test kit that blood group changes that is used for of claim 1 that comprises.

Images