CN102516373A - Pneumococcal surface protein A without human homology, purification method and application thereof - Google Patents
Pneumococcal surface protein A without human homology, purification method and application thereof Download PDFInfo
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Abstract
The invention discloses pneumococcal surface protein A without human homology, a purification method and application thereof. The pneumococcal surface protein A without human homology is expressed by an amino acid sequence shown in a sequence table SEQ ID No.1 or an amino acid sequence shown in a sequence table SEQ ID No.2. Antigenicity and protectiveness of the pneumococcal surface protein A without human homology are not lower than those of the wild type pneumococcal surface protein A (RX-1 subtype), and the homology between the pneumococcal surface protein A (RX-1 subtype) and the human cardiac muscle myosin is decreased, and accordingly the pneumococcal surface protein A without human homology can be applied to development of vaccine safely.
Description
Technical field
The present invention relates to the pneumococcal surface protein A of removal people homology of the partial sequence replacement of a kind of pneumococcal surface protein A, express purification process and purposes.
Background is described
Streptococcus pneumoniae (Streptococcus pneumoniae) is claimed streptococcus pneumoniae again, is encapsulated Gram-positive diplococcus.The polysaccharide pod membrane is main virulence factor, according to the composition difference of pod membrane, has identified about 90 different streptococcus pneumoniae serotypes.Streptococcus pneumoniae can cause diseases such as pneumonia, meningitis, septicemia, otitis media, and sickness rate is the highest in infant below 2 years old and the elderly, has become and has caused one of dead major reason in the world wide.Estimated according to WHO that the whole world had 1,600,000 people to die from all kinds of streptococcus pneumoniae diseases every year in 2005, comprising the children of 70-100 below 10,000 years old, most deaths occur in developing country.In China, pneumonia also is to cause one of dead No.1 killer of infant.HIV infection and the other diseases relevant with immunodeficient have increased the possibility of pneumonia infection coccus disease greatly.The streptococcus pneumoniae resistance problem that increases day by day also makes the exploitation vaccine become more urgent with control streptococcus pneumoniae property disease.Current, Influenza A H1N1 is wreaked havoc in the whole world, often causes great mortality ratio by the respiratory tract accompanying infection pneumonia of its initiation, and national governments are advocating the inoculation Pnu-Imune 23 when dropping into these influenza vaccines of exploitation energetically.In order to reduce pneumococcal infection and carrying rate, ensure people ' s health, prevention and containment illness spread, the inoculation Pnu-Imune 23 is very necessary.WHO includes pneumococcal conjugated vaccine in national Immunization programme in suggestion developing country in 2007.
The Pnu-Imune 23 product of supply is mainly polysaccharide vaccine and combined vaccine in the market.Wherein the antigen of 23 valency pneumococcal capsular polysaccharide vaccines is the thymus gland independent form, a little less than the immune effect to infant and the elderly, has been mainly used in high risk big-age-child of streptococcus pneumoniae property disease and grownup, can not be used for the children below 2 years old.The protein-polysaccharide combined vaccine is like 7 valency pneumonia combined vaccines (PCV-7); Immunogenicity in each age group is all very strong; But only approval is used for children below 5 years old at present; And 7 serotypes of its covering only account for Asia area 55% of the serotype of causing a disease, and make the replacement that possibly occur serotype in the application process.860 yuan/the agent of home market price of PCV-7 at present, child below 2 years old need inoculate 3-4 time, and price is higher.Pneumovax market is huge both at home and abroad, and efficiently and cheaply vaccine is imperative for development of new, is stepping up to develop recombinant protein vaccine and DNA new generation vaccine both at home and abroad.The exploitation of recombinant protein vaccine mainly concentrates on the important candidate albumens such as PspA, PsaA and hemolysin, and wherein PspA is potentialization and studies one of maximum candidate albumen.
PspA, streptococcus pneumoniae surface protein A extensively is present in 90 kinds of streptococcus pneumoniae serotypes, and the streptococcus pneumoniae PspA protein molecular quality of different serotypes is different, between 60～100Ka.The PspA molecule can be divided into two extended familys, five subgroups (clade).PspA mainly is made up of five territories: hold the C end to be divided into signal peptide, superhelix district, Histidine enrichment region, choline binding district and C end tail successively from N.The superhelix district is an important antigen determining zone.Hold at N in the site of monoclonal antibody identification, and this district has highly variable.In different serotypes, the C of PspA end hydrophobic region has conserved structure.It is to combine to make it loss of function with Lf lactoferrin and suppress complement activation that PspA mainly acts on, thereby reduces C3b disturbs complement-mediated in the deposition on streptococcus pneumoniae surface the opsonization of engulfing.This albumen has immunogenicity efficiently, research proof in mouse, and PspA albumen can excite the protective immunological reaction to pneumococcal infection.
The PspA vaccine is researched and developed in Sanofi Pasteur (Sai Nuofei-pasteur) and University of Alabama at Birmingham (UAB) cooperation, and has accomplished the clinical first phase experiment of two vaccine products, and result's proof has extraordinary provide protection.But, also observe the of short duration rising of antibody of anti-human body myocardium myo sphaeroprotein simultaneously on one's body part test person adopting PspA (RX-1 hypotype) when making an experiment as protective antigen.Anti-myocardium protein antibody normal presence in human body; Show that anti-myocardium myo globulin antibody can cause the clinical evidence of disease even lack at present; But still there is potential safety hazard, the application of PspA vaccine is brought huge obstacle, reduce very necessity of PspA and myocardium protein cross reaction.
Summary of the invention
The objective of the invention is to overcome wild-type pneumonia surface protein A as antigenic deficiency, a kind of pneumococcal surface protein A of the people's of removal homology is provided.
Second purpose of the present invention provides a kind of purposes of expressing the pneumococcal surface protein A that removes people's homology.
The 3rd purpose of the present invention provides a kind of gene of expressing the pneumococcal surface protein A that removes people's homology.
The 4th purpose of the present invention provides a kind of pneumococcal surface protein A gene recombined vector that contains the expressing human homology.
The 5th purpose of the present invention provides a kind of bacterial strain of pneumococcal surface protein A gene recombined vector of the people's of containing homology.
The 6th purpose of the present invention provides the purification process of a kind of pneumococcal surface protein A of the people's of removal homology.
Technical scheme of the present invention is summarized as follows:
Removing the pneumococcal surface protein A of people's homology, is the aminoacid sequence shown in aminoacid sequence shown in the sequence table SEQ ID No.1 or the sequence table SEQ ID No.2.
The application of the pneumococcal surface protein A of above-mentioned removal people homology in the vaccine of preparation prevention streptococcus pneumoniae infection.
Express the gene of the pneumococcal surface protein A that removes people's homology, the gene that is the aminoacid sequence shown in the said expressed sequence table SEQ ID No.1 is with shown in the sequence table SEQ ID No.3; The gene of the aminoacid sequence shown in the said expressed sequence table SEQ ID No.2 is with shown in the sequence table SEQ ID No.4.
Contain and express the pneumococcal surface protein A gene recombined vector of removing people's homology, gene shown in the sequence table SEQ ID No.3 is inserted into obtains recombinant vectors 1 among the expression vector PET9a; Or gene shown in the sequence table SEQ ID No.4 is inserted into obtains recombinant vectors 2 among the expression vector PET9a.
The bacterial strain that contains the pneumococcal surface protein A gene recombined vector of removing people's homology imports to said recombinant vectors 1 among the intestinal bacteria E.Coli BL21 or said recombinant vectors 2 is imported to the bacterial strain that obtains containing the pneumococcal surface protein A gene recombined vector of removing people's homology among the intestinal bacteria E.Coli BL21.
Remove the purification process of the pneumococcal surface protein A of people's homology, comprise the steps:
(1) fermentation contains the bacterial strain of the pneumococcal surface protein A gene recombined vector of removing people's homology;
(2) with centrifugal 15 minutes of bacterial strain fermentation liquor 7200rpm, collecting precipitation, with 20 mmoles of pH=8.0/rise the resuspended deposition of tris, homogenate cytoclasis, centrifugal 30 minutes of 8000rpm gets supernatant;
(3) batch absorption: the supernatant that Q sepharose fast flow and step (2) are obtained mixed whip attachment 2 hours, and buffer system is 20 mmoles/the rise tris of pH=8.0; 20 mmoles of the pH=5.4 of the supernatant volume that obtains with the doubly said step of 3-5 (2)/rise the citric acid-sodium citrate damping fluid with the adsorbed product wash-out; With 3-5 times of sample volume contain the 350-450 mmole/liter sodium-chlor pH=5.4 20 mmoles/liter the citric acid-sodium citrate buffer solution elution, obtain the Elute product;
(4) with the Elute product with 20 mmoles of pH=5.4/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(5) the Q post is prepared: with 20 mmoles of the pH=5.4 of 3-5 column volume doubly/rise citric acid-sodium citrate damping fluid balance Q post;
(6) go up appearance: appearance Q post on the product that step (4) is obtained;
(7) with 20 mmoles of the pH=5.4 that contains 50-150 mmole sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 160-300 mmole/the rise pH=5.4 of sodium-chlor of 3-5 times of column volume/rise Hydrocerol A lemon-lemon acid sodium damping fluid Elute wash-out;
(8) product that step (7) is obtained is with 20 mmoles of pH=4.0/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(9) the SP post is prepared: with 20 mmoles of the pH=4.0 of 3-5 column volume doubly/rise citric acid-sodium citrate damping fluid balance SP post;
(10) go up appearance: with appearance SP post on the product of step (8) acquisition;
(11) with 20 mmoles that contain 250-350 mmole/the rise pH=4.0 of sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 450-550 mmole/the rise pH=4.0 of sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid Elute wash-out; Obtain the pneumococcal surface protein of the removal people homology of purity more than 90%.
Advantage of the present invention:
A kind of pneumococcal surface protein A of the people's of removal homology has antigenicity and the protectiveness that is not weaker than wild-type pneumococcal surface protein A (RX-1 hypotype), and has reduced the homology of pneumococcal surface protein A (RX-1 hypotype) and human body myocardium myo sphaeroprotein.Make the developing vaccines that is applied to of its safety.
Description of drawings
Fig. 1 is 16 amino acid that derive from the pneumonia different strains; Reach 16 amino acid that have high homology with people's cardiac muscle myosin in wild-type pneumonia surface protein (RX-1 hypotype) sequence, the mutant strain of the new pneumonia surface protein (RX-1 hypotype) of formation.Then with mutant strain and wild-type self sequence, through the similar software analysis of Paircoil2, the possibility of its 16 α-Luo Xuanjiegous that amino acid forms is compared for the structure that forms, and it analyzes collection of illustrative plates.
A: wild-type pneumonia surface protein PspA-RX-1, B: pneumonia surface protein PspA-RX-1-BG9739 (SEQ ID No.1), C: pneumonia surface protein PspA-RX-1-BG3296 (SEQ ID No.2), D: pneumonia surface protein PspA-RX-1-self.
Fig. 2 is the SDS-PAGE electrophorogram of pneumonia surface protein A (PspA-RX-1, PspA-RX-1-BG9739 (Mutant1), PspA-RX-1-BG3296 (Mutant2)) purifying.
1 is the standard protein molecular weight from right to left; 2 is PspA-RX-1; 3 is PspA-RX-1-BG9739; 4 is PspA-RX-1-BG3296
Fig. 3 is mouse immune pneumonia surface protein PspA-RX-1, PspA-RX-1-BG9739, PspA-RX-1-BG3296 and the antibody horizontal that produces.
Fig. 4 is immune pneumonia surface protein PspA-RX-1, PspA-RX-1-BG9739, and the mouse of PspA-RX-1-BG3296 is attacked the protection ratio of test through the pneumonia streptococcus bacterium.
Embodiment
Below in conjunction with specific embodiment the present invention is further described.
1., confirmed in the PspA-RX-1 subtype protein that through the sequence homology software analysis 16 aminoacid sequences and one section sequence of people's cardiac muscle myosin have high homology, like this if as the antigenic words of immunity, there is immune potential safety hazard in use PspA-RX-1.These 16 aminoacid sequences are EAKAKLEEAEKKATEA
2.; Adopt the structure of these 16 aminoacid sequences of the similar software analysis of Paircoil2, and from the PspA molecule of other hypotypes, pick out 16 different amino acid fragments 16 aminoacid sequences of the PspA-RX-1 of wild-type are replaced, form new PspA-RX-1 sequence; Be PspA-RX-1-BG9739; PspA-RX-1-BG3296 through the simulation of molecule secondary structure, predicts the structure contrast of mimic structure and wild-type PspA-RX-1 again; We have selected the mutant strain PspA-RX-1-BG9739 of two PspA-RX-1s minimum with respect to wild-type PspA-RX-1 structural changes, PspA-RX-1-BG3296
3., 16 amino acid EAKAKLEEAEKKATEA in the PspA-RX-1 wild-type sequence are replaced by EAEKKVTEARQKLDAE (deriving from streptococcus pneumoniae BG9739) and EALQNKLAAKKAELAKK (deriving from streptococcus pneumoniae BG3296) respectively.
Obtain: remove the pneumococcal surface protein A of people's homology, respectively with the aminoacid sequence shown in the sequence table SEQ ID No.1 or with the aminoacid sequence shown in the sequence table SEQ ID No.2.
Express the gene of the pneumococcal surface protein A that removes people's homology, the gene that is the aminoacid sequence shown in the said expressed sequence table SEQ ID No.1 is with shown in the sequence table SEQ ID No.3; The gene of the aminoacid sequence shown in the said expressed sequence table SEQ ID No.2 is with shown in the sequence table SEQ ID No.4.
The dna sequence dna that to be the PspA-RX-1-BG9739 gene optimized with DNA2.0 of gene shown in the SEQ ID No.3;
The dna sequence dna that to be the PspA-RX-1-BG3296 gene optimized with DNA2.0 of gene shown in the SEQ ID No.4.
Contain and express the pneumococcal surface protein A gene recombined vector of removing people's homology, gene shown in the sequence table SEQ ID No.3 is inserted into obtains recombinant vectors 1 among the expression vector PET9a; Or gene shown in the sequence table SEQ ID No.4 is inserted into obtains recombinant vectors 2 among the expression vector PET9a.
The bacterial strain that contains the pneumococcal surface protein A gene recombined vector of removing people's homology imports to said recombinant vectors 1 among the intestinal bacteria E.Coli BL21 or said recombinant vectors 2 is imported to the bacterial strain that obtains containing the pneumococcal surface protein A gene recombined vector of removing people's homology among the intestinal bacteria E.Coli BL21.
Recombinant expressed in the BL21 intestinal bacteria.After adding IPTG, target protein is expressed in the tenuigenin.
Embodiment 5
Remove the purification process (SEQ ID No.1, SEQ ID No.2 are the method purifying that adopts present embodiment respectively) of the pneumococcal surface protein A of people's homology, comprise the steps:
(1) bacterial strain of the pneumococcal surface protein A gene recombined vector that contains people's homology of fermentation embodiment 4 preparations;
(2) with centrifugal 15 minutes of said bacterial strain fermentation liquor 7200rpm, collecting precipitation, with 20 mmoles of pH=8.0/rise the resuspended deposition of tris, homogenate cytoclasis, centrifugal 30 minutes of 8000rpm gets supernatant;
(3) batch absorption: the supernatant that Q sepharose fast flow and step (2) are obtained mixed whip attachment 2 hours, and buffer system is 20 mmoles/the rise tris of pH=8.0; 20 mmoles of the pH=5.4 of the supernatant volume that obtains with 4 times of said steps (2)/rise Hydrocerol A lemon-lemon acid sodium damping fluid with the adsorbed product wash-out; With 20 mmoles that contain 400 mmoles/the rise pH=5.4 of sodium-chlor of 4 times of sample volumes/rise the citric acid-sodium citrate buffer solution elution, obtain the Elute product;
(4) with the Elute product with 20 mmoles of pH=5.4/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(5) the Q post is prepared: with 20 mmoles of the pH=5.4 of 4 times column volumes/rise citric acid-sodium citrate damping fluid balance Q post;
(6) go up appearance: appearance Q post on the product that step (4) is obtained;
(7) with 20 mmoles of the pH=5.4 that contains 100 mmole sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 250 mmoles/the rise pH=5.4 of sodium-chlor of 4 times of column volumes/rise Hydrocerol A lemon-lemon acid sodium damping fluid Elute wash-out;
(8) product that step (7) is obtained is with 20 mmoles of pH=4.0/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(9) the SP post is prepared: with 20 mmoles of the pH=4.0 of 4 times column volumes/rise citric acid-sodium citrate damping fluid balance SP post;
(10) go up appearance: with appearance SP post on the product of step (8) acquisition;
(11) with 20 mmoles that contain 300 mmoles/the rise pH=4.0 of sodium-chlor of 4 times of column volumes/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 500 mmoles/the rise pH=4.0 of sodium-chlor of 4 times of column volumes/rise citric acid-sodium citrate damping fluid Elute wash-out; Obtain the pneumococcal surface protein of the removal people homology of purity more than 90%.(Fig. 2).
Embodiment 6 is with PspA-RX-1, and PspA-RX-1-BG9739 (SEQ ID No.1) and each 50ug of PspA-RX-1-BG3296 (SEQ ID No.2) carry out immunity to mouse.Each immunity was twice in 0,14 day.With phosphagel phosphaljel adjuvant negative control the most.Carry out antibody blood sampling in 28 days and measure, and carry out intravenous injection with the streptococcus pneumoniae of 500CFU.The survival ability of monitoring mouse.Its antibody horizontal Fig. 3.This data presentation is carried out immunity with PspA-RX-1-BG9739 and PspA-RX-1-BG3296, can produce the antibody of Anti-RX-1, and its level is not less than the level of carrying out immunity with wild-type RX-1 antigen on statistics.
The fate of mouse survival is shown in Figure 4.Demonstrate Mutant1 and have the protection identical with the RX-1 wild-type protein with Mutant 2.And the phosphagel phosphaljel adjuvant does not have protection.
We have found the polypeptide fragment of 16 amino acid lengths in the PspA-RX-1 subtype protein.Its sequence is: EAKAKLEEAEKKATEA.The sequence of this sequence and people's myocardium myo sphaeroprotein has highly similar.
16 amino acid with high homology that the present invention is directed to wild-type PspA-RX-1 antigenic determinant and human body myocardium myo sphaeroprotein are guaranteeing to carry out the amino acid replacement under the minimum situation of structural changes influence.Two molecules of new production have antigenicity and the protectiveness that is not weaker than wild-type PspA-RX-1, and have reduced PspA-RX-1 and the proteic homology of people.Make the developing vaccines that is applied to of its safety.
We pick out the simulation that 16 different amino acid fragments carry out the molecule secondary structure and (adopt the similar software of Paircoil2, Fig. 1) from the PspA molecule of other hypotypes.The contrast prediction result, we have selected the mutant strain of two minimum PspA-RX1 of structural changes.Wherein 16 amino acid EAKAKLEEAEKKATEA in the PspA wild-type sequence are replaced by EAEKKVTEARQKLDAE (deriving from streptococcus pneumoniae BG9739) and EALQNKLAAKKAELAKK (deriving from streptococcus pneumoniae BG3296) respectively.
We are through cooperating with UAB university; With PspA-RX-1; PspA-RX-1-BG9739 (SEQ ID No.1) and PspA-RX-1-BG3296 (SEQ ID No.2) carry out the test of animal immune property, find that later two the sudden change PspA-RX-1-BG9739 (SEQ ID No.1) that form of 16 amino acid of replacement have identical poison with PspA-RX-1-BG3296 (SEQ ID No.2) with wild-type PspA-RX-1 and attack protectiveness
Embodiment 7
Remove the purification process of the pneumococcal surface protein A of people's homology, comprise the steps:
(1) bacterial strain of the pneumococcal surface protein A gene recombined vector that contains people's homology of fermentation embodiment 4 preparations;
(2) with centrifugal 15 minutes of said bacterial strain fermentation liquor 7200rpm, collecting precipitation, with 20 mmoles of pH=8.0/rise the resuspended deposition of tris, homogenate cytoclasis, centrifugal 30 minutes of 8000rpm gets supernatant;
(3) batch absorption: the supernatant that Q sepharose fast flow and step (2) are obtained mixed whip attachment 2 hours, and buffer system is 20 mmoles/the rise tris of pH=8.0; 20 mmoles of the pH=5.4 of the supernatant volume that obtains with 3 times of said steps (2)/rise Hydrocerol A lemon-lemon acid sodium damping fluid with the adsorbed product wash-out; With 20 mmoles that contain 350 mmoles/the rise pH=5.4 of sodium-chlor of 3 times of sample volumes/rise the citric acid-sodium citrate buffer solution elution, obtain the Elute product;
(4) with the Elute product with 20 mmoles of pH=5.4/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(5) the Q post is prepared: with 20 mmoles of the pH=5.4 of 3 times column volumes/rise citric acid-sodium citrate damping fluid balance Q post;
(6) go up appearance: appearance Q post on the product that step (4) is obtained;
(7) with 20 mmoles of the pH=5.4 that contains 50 mmole sodium-chlor of 3 times of column volumes/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 160 mmoles/the rise pH=5.4 of sodium-chlor of 3 times of column volumes/rise Hydrocerol A lemon-lemon acid sodium damping fluid Elute wash-out;
(8) product that step (7) is obtained is with 20 mmoles of pH=4.0/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(9) the SP post is prepared: with 20 mmoles of the pH=4.0 of 3 times column volumes/rise citric acid-sodium citrate damping fluid balance SP post;
(10) go up appearance: with appearance SP post on the product of step (8) acquisition;
(11) with 20 mmoles that contain 250 mmoles/the rise pH=4.0 of sodium-chlor of 3 times of column volumes/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 450 mmoles/the rise pH=4.0 of sodium-chlor of 3 times of column volumes/rise citric acid-sodium citrate damping fluid Elute wash-out; Obtain the pneumococcal surface protein of the removal people homology of purity more than 90%.
Embodiment 8
Remove the purification process of the pneumococcal surface protein A of people's homology, comprise the steps:
(1) bacterial strain of the pneumococcal surface protein A gene recombined vector that contains people's homology of fermentation embodiment 4 preparations;
(2) with centrifugal 15 minutes of said bacterial strain fermentation liquor 7200rpm, collecting precipitation, with 20 mmoles of pH=8.0/rise the resuspended deposition of tris, homogenate cytoclasis, centrifugal 30 minutes of 8000rpm gets supernatant;
(3) batch absorption: the supernatant that Q sepharose fast flow and step (2) are obtained mixed whip attachment 2 hours, and buffer system is 20 mmoles/the rise tris of pH=8.0; 20 mmoles of the pH=5.4 of the supernatant volume that obtains with 5 times of said steps (2)/rise Hydrocerol A lemon-lemon acid sodium damping fluid with the adsorbed product wash-out; With 20 mmoles that contain 450 mmoles/the rise pH=5.4 of sodium-chlor of 5 times of sample volumes/rise the citric acid-sodium citrate buffer solution elution, obtain the Elute product;
(4) with the Elute product with 20 mmoles of pH=5.4/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(5) the Q post is prepared: with 20 mmoles of the pH=5.4 of 5 times column volumes/rise citric acid-sodium citrate damping fluid balance Q post;
(6) go up appearance: appearance Q post on the product that step (4) is obtained;
(7) with 20 mmoles of the pH=5.4 that contains 150 mmole sodium-chlor of 5 times of column volumes/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 300 mmoles/the rise pH=5.4 of sodium-chlor of 5 times of column volumes/rise Hydrocerol A lemon-lemon acid sodium damping fluid Elute wash-out;
(8) product that step (7) is obtained is with 20 mmoles of pH=4.0/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(9) the SP post is prepared: with 20 mmoles of the pH=4.0 of 5 times column volumes/rise citric acid-sodium citrate damping fluid balance SP post;
(10) go up appearance: with appearance SP post on the product of step (8) acquisition;
(11) with 20 mmoles that contain 350 mmoles/the rise pH=4.0 of sodium-chlor of 5 times of column volumes/rise citric acid-sodium citrate damping fluid wash wash-out, again with 20 mmoles that contain 550 mmoles/the rise pH=4.0 of sodium-chlor of 5 times of column volumes/rise citric acid-sodium citrate damping fluid Elute wash-out; Obtain the pneumococcal surface protein of the removal people homology of purity more than 90%.
Claims (6)
1. remove the pneumococcal surface protein A of people's homology, it is characterized in that the pneumococcal surface protein A of said removal people homology is the aminoacid sequence shown in aminoacid sequence shown in the sequence table SEQ ID No.1 or the sequence table SEQ ID No.2.
2. the pneumococcal surface protein A of the removal people homology of claim 1 is in the application of the vaccine of preparation prevention streptococcus pneumoniae infection.
3. express the gene of the pneumococcal surface protein A of the said removal of claim 1 people homology, the gene that it is characterized in that the aminoacid sequence shown in the said expressed sequence table SEQ ID No.1 is with shown in the sequence table SEQ ID No.3; The gene of the aminoacid sequence shown in the said expressed sequence table SEQ ID No.2 is with shown in the sequence table SEQ ID No.4.
4. the pneumococcal surface protein A gene recombined vector that contains the expressing human homology is characterized in that gene shown in the sequence table SEQ ID No.3 is inserted into and obtains recombinant vectors 1 among the expression vector PET9a; Or gene shown in the sequence table SEQ ID No.4 is inserted into obtains recombinant vectors 2 among the expression vector PET9a.
5. the bacterial strain that contains the pneumococcal surface protein A gene recombined vector of people's homology is characterized in that said recombinant vectors 1 is imported among the intestinal bacteria E.Coli BL21 or said recombinant vectors 2 imported to the bacterial strain of the pneumococcal surface protein A gene recombined vector that obtains containing people's homology among the intestinal bacteria E.Coli BL21.
6. remove the purification process of the pneumococcal surface protein A of people's homology, it is characterized in that comprising the steps:
(1) bacterial strain of the pneumococcal surface protein A gene recombined vector that contains people's homology of fermentation claim 4 preparation;
(2) with centrifugal 15 minutes of said bacterial strain fermentation liquor 7200rpm, collecting precipitation, with pH=8.0 20 mmoles/rise the resuspended deposition of tris, homogenate cytoclasis, centrifugal 30 minutes of 8000rpm gets supernatant;
(3) batch absorption: the supernatant that Q sepharose fast flow and step (2) are obtained mixed whip attachment 2 hours, and buffer system is 20 mmoles/the rise tris of pH=8.0; The concentration of the pH=5.4 of the supernatant volume that obtains with the doubly said step of 3-5 (2) be 20 mmoles/liter the citric acid-sodium citrate damping fluid with the adsorbed product wash-out; With 3-5 times of sample volume contain the 350-450 mmole/liter the concentration of sodium-chlor pH=5.4 be 20 mmoles/liter the citric acid-sodium citrate buffer solution elution, obtain the Elute product;
(4) with the Elute product with 20 mmoles of pH=5.4/liter citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(5) the Q post is prepared: with 20 mmoles of the pH=5.4 of 3-5 column volume doubly/liter citric acid-sodium citrate damping fluid balance Q post;
(6) go up appearance: appearance Q post on the product that step (4) is obtained;
(7) with 20 mmoles of the pH=5.4 that contains 50-150 mmole sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid wash wash-out, again with pH=5.4 20 mmoles that contain 160-300 mmole/rise sodium-chlor of 3-5 times of column volume/rise Hydrocerol A lemon-lemon acid sodium damping fluid Elute wash-out;
(8) product that step (7) is obtained is with pH=4.0 20 mmoles/rise citric acid-sodium citrate damping fluid ultrafiltration desalting treatment;
(9) the SP post is prepared: with pH=4.0 20 mmoles of 3-5 column volume doubly/rise citric acid-sodium citrate damping fluid balance SP post;
(10) go up appearance: with appearance SP post on the product of step (8) acquisition;
(11) with pH=4.0 20 mmoles that contain 250-350 mmole/rise sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid wash wash-out, again with pH=4.0 20 mmoles that contain 450-550 mmole/rise sodium-chlor of 3-5 times of column volume/rise citric acid-sodium citrate damping fluid Elute wash-out; Obtain the pneumococcal surface protein of the removal people homology of purity more than 90%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104306965A (en) * | 2014-10-31 | 2015-01-28 | 天津康希诺生物技术有限公司 | Immunogenic composition for preventing streptococcus pneumoniae infectious diseases and preparation method thereof |
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| EP1993601B1 (en) * | 2006-02-02 | 2011-12-07 | University Of Alabama Research Foundation | Non-coiled protective regions of pneumococcal surface proteins pspa and pspc |
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| US5965141A (en) * | 1991-02-15 | 1999-10-12 | Uab Research Foundation | Epitopic regions of pneumococcal surface protein a |
| US6592876B1 (en) * | 1993-04-20 | 2003-07-15 | Uab Research Foundation | Pneumococcal genes, portions thereof, expression products therefrom, and uses of such genes, portions and products |
| WO2006127020A1 (en) * | 2005-05-19 | 2006-11-30 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention | FUNCTIONAL EPITOPES OF STREPTOCOCCUS PNEUMONIAE PsaA ANTIGEN AND USES THEREOF |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104306965A (en) * | 2014-10-31 | 2015-01-28 | 天津康希诺生物技术有限公司 | Immunogenic composition for preventing streptococcus pneumoniae infectious diseases and preparation method thereof |
| WO2016066095A1 (en) * | 2014-10-31 | 2016-05-06 | 天津康希诺生物技术有限公司 | Immunogenic composition for preventing streptococcus pneumoniae infectious diseases and preparation method thereof |
| CN104306965B (en) * | 2014-10-31 | 2017-05-10 | 康希诺生物股份公司 | Immunogenic composition for preventing streptococcus pneumoniae infectious diseases and preparation method thereof |
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