CN104096223A

CN104096223A - Method for enhancing 13-valent pneumococcal polysaccharide protein bonder immunogenicity

Info

Publication number: CN104096223A
Application number: CN201410198752.3A
Authority: CN
Inventors: 李建平
Original assignee: JIANGSU KANGTAI BIOMEDICAL TECHNOLOGY CO LTD
Current assignee: JIANGSU KANGTAI BIOMEDICAL TECHNOLOGY CO LTD
Priority date: 2014-05-11
Filing date: 2014-05-11
Publication date: 2014-10-15
Anticipated expiration: 2034-05-11
Also published as: CN104096223B

Abstract

The invention discloses a method for enhancing 13-valent pneumococcal polysaccharide protein bonder immunogenicity, and the method is as follows: adding all-powerful epitope peptide (P30) into CRM197A (A chain of diphtheria toxin variant 197), using genetic recombinant Escherichia coli to produce a P30-containing P30CRM197A; and connecting 13 different serotype polysaccharides (comprising 1, 3, 4, 5, 6 A, 6 A, 7 F, 9 V, 14, 18C, 19 A, 19F and 23 F ) to the P30CRM197A protein carrier by covalent bonds to form a 13-valent pneumococcal polysaccharide-P30CRM197A bonder; compared with a 13-valent pneumococcal polysaccharide-CRM197A bonder obtained by a corresponding protein carrier CRM197A which does not contain the all-powerful epitope peptide (P30), the immunogenicity of the 13-valent pneumococcal polysaccharide-P30CRM197A bonder obtained by the method is increased by 3-5 times than that of a contrast.

Description

A kind of immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates

Technical field

The present invention relates to a kind of immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates.

Background technology

When polysaccharide is when being covalently linked on protein carrier, haptenic polysaccharide can be transformed into holoantigen, the immunogenicity of polysaccharide is enhanced.With the synthetic Duo Tang of the method ?protein binding vaccine be widely used in children's, successfully prevented to comprise the infection of the antibacterials such as streptococcus pneumoniae, epidemic cerebrospinal meningitis coccus and popular haemophilus b type.

For the synthesis of the protein carrier of GL-PP conjugate, have multiple, the popular haemophilus surface protein D producing as tetanus toxoid, diphtheria toxoid, diphtheria toxin, diphtherotoxin variant CRM197 and gene recombination technology etc.; But, due to the immunological characteristic of different albumen, variant with the immunogenicity of the synthetic GL-PP conjugate of different protein carriers, after animal body immunity, for synthesizing by different protein carriers and same polysaccharide the GL-PP conjugate forming, the polysaccharide immunogenic manifesting is also different.As can be seen here, the protein carrier that adopts different technologies to produce, the immunogenicity of the GL-PP conjugate being synthesized is variant.

After entering in animal body, antigen is processed cell (Antigen Process Cell through antigen, APC) process the rear epitope peptide (epitope) [1] that produces, with ajor histocompatibility complex (Major Histocompatibility Complex, MHC) after molecule combination, and then be presented on APC cell surface, and can be identified by T lymphocyte, this is the conclusion that immunology has drawn by experiment.It is now know that, and the immunogenicity of a known epitope peptide depends on three factors:

The first, suitably the generation of epitope peptide;

Second and the presenting of the Major histocompatibility complex molecule of epitope peptide combination;

Presenting of the T cell of the conjugate that three, identification epitope peptide and ajor histocompatibility complex form.

Wherein, lacking of any one link, all will cause immunne response disappearance.

With the test that mice carries out, show, the conjugate molecule that lacks suitable epitope peptide and the formation of ajor histocompatibility complex is the reason that the most often causes animal body immune response disappearance.Ajor histocompatibility complex has the multiform state property of height, and known epitope peptide only just can be bonded to ajor histocompatibility complex by one or several allele (Alleles), rather than whole epitope peptide fragment.In addition, have experimental result to show, due to antigen, process inappropriately, or T cell tolerance vacancy, also can cause the disappearance of immune response.

There is experiment to show, the epitope peptide FNNFTVSFWLRVPKVSASHLE (being called P30) [2] of tetanus toxin, can with a large amount of different ajor histocompatibility complex Class II combinations, show that it can be by T cell recognition, there is omnipotent immunogenic characteristic, be termed omnipotent epitope peptide.

Omnipotent immunogenicity epitope peptide has homotype and the shaped body combination with a plurality of mankind's major histocompatibility antigen complex Class II molecules.This omnipotent epitope peptide and the random combination of mankind's major histocompatibility antigen complex Class II molecule can be used for developing synthetic vaccine, because the replying of the acquired immune system of its major part individuality in can activation crowd.

Research shows, P30 epitope peptide by tetanus toxin 947 ?967 aminoacid sequences form (FNNFTVSFWLRVPKVSASHLE).At the albumen that contains this epitope peptide, enter after body, albumen will be ingested to APC cell, digested degraded.But these epitope peptides will be preserved complete, with ajor histocompatibility complex in conjunction with after, be present in APC cell surface, and by T cell recognition, this show omnipotent epitope peptide in an identical manner with multiple DR molecular action.

MHC molecule is the polygamy receptor of antigen after processing, and its function is in the process of tolerance induction in thymus and periphery immune response exotic antigen, presents the epitope peptide in its antigen.Therefore, MHC molecule must present a large amount of epitope peptide (allow better antigen recognition, but more consume T cell deposit), and reaches balance in a little epitope peptide (a large amount of T cells is laid in, but a little presents exotic antigen effectively).That is to say, if contain in antigen and can preserve integrity, and representative a small amount of epitope peptide after APC cell processed, with MHC in conjunction with after, just can stimulate a certain amount of T cell, set up immunological memory effect, and this process can not consume excessive T cell, to avoid consuming a large amount of T cells, cause immunologic tolerance.The antigen that contains this epitope peptide has stronger immunogenicity, and this has also just explained why tetanus toxoid has very strong immunogenic reason.

The stimulation epitope peptide of most of T cell of now finding acts on limited for different MHC Class II monomers (haplotype), different animals is preserved and presented different antigen polypeptide regions (epitopes) stimulates its T cell.The gene restriction of this T cell-stimulating activity has hindered with synthetic method and has carried out vaccine development, and this method is for crowds' different on gene application, should be unusual effective method.Those are found to have stimulates the T cytositimulation epitope peptide that multiple mice is individual and/or be associated with most of human body MHC Class II molecules, and the effective way of an omnipotent activating T cell of design is provided.In common proteantigen, add omnipotent epitope peptide (also referred to as omnipotent T cellular antigens bunch) P30, can be by the MHC Class II molecular recognition of most animals.This T cellular antigens bunch can be used in direct inducing T cell, or the B cells produce of offering help is directed to weak immunogenic antibody, the effect of enhancing body Acquired immune response.

Pathogenic bacteria can be expressed high molecular conventionally, and what be wrapped in bacterium surface is capsular polysaccharide, is called for short polysaccharide.For adult, capsular polysaccharide is to have good immunogenicity antigen, can be used for preparing vaccine; But, for children's, 2 years old following infant, it is the non-T of depending on cellular antigens that capsular polysaccharide is considered to.Experiment demonstration, when being used as antigen, the response that capsular polysaccharide can be induced wild strain or T cell disappearance mice produces polysaccharide specific IgM antibodies; But, do not induce IgM antibody to the conversion of IgG antibody.Human experimentation also shows, as vaccine, polysaccharide can induce adult to produce protection antibody, but cannot induce the immunne response of infant; That is to say, children's, repeat after immune capsular polysaccharide antigen, without the response of antibody enhancing for the second time, T cell memory that also cannot be inducing sustained.

Modern immunological experiment confirms, the advantage of GL-PP combined vaccine and the comparison of holosaccharide vaccine is, the former can induction of immunity response.When the capsular polysaccharide covalent bond of non-dependence T cell be connected to carrier protein and the GL-PP conjugate that forms, in immunity after mammal, can help the IgG antibody that B cell produces the polysaccharide part being directed in conjugate by inducing T cell.Therefore, GL-PP conjugate induction polysaccharide specific antibody IgM is converted into IgG, the differentiation of memory B cell, and long-standing T cell memory.

Streptococcus pneumoniae, also claim streptococcus pneumoniae, worldwide to cause the mankind to fall ill and one of dead main pathogenic bacteria, especially in infant, chronic heart and lung diseases patient, old people and immunologic hypofunction person, easily cause diseases such as bacterial pneumonia, meningitis, bacteremia and acute otitis media.According to World Health Organization (WHO) (WHO) report on Epidemiological in 1999, estimate the various diseases that has at least every year 1000000 years old following infant and child to die of pneumonia due to coccus.According to industrially developed country, the sickness rate of the invasive pneumococcal pneumonia of 2 years old Infants Below is up to 160 cases in every 100,000 people; In American-European countries, the bacterial meningitis of 25%-40% is caused by streptococcus pneumoniae.The U.S. approximately has 150,000～570,000 routine pneumococcal pneumonias every year, 2600～6200 routine pneumococcal meningitis, and both cause 40,000 people dead altogether every year; The bacteremic sickness rate of streptococcus pneumoniae is in 15,/10 ten thousand～19,/10 ten thousand left and right, case fatality rate approximately 25%～30%.In addition, have 50%～67% for due to streptococcus pneumoniae in bacillary otitis media, be often difficult to radical cure, relapse rate is high, affects child's healthy growth and development.According to statistics, the annual infant of the Jin U.S. is just gone to see a doctor up to seven million peoples time because of otitis media, to medical system, has brought heavy burden.Therefore, when septivalency pneumonia polysaccharide conjugate vaccine is succeeded in developing in U.S.'s Hui Shi pharmacy, and obtain after U.S. FDA production and sales licence, the infectious disease committee of AAP and U.S. immunity test consultative committee inoculate this vaccine in the child in the 2-4 year of advising immediately at the beginning of 2000 at the infant below 2 years old and hypoimmunity.

Multinomial pneumococcal Epidemiological study result shows, different countries and regions, and pneumococcal popular bacterial strain is that serotype is different.The 7 valency pneumococcal Polysaccharide Conjugate Vaccines of U.S. Wyeth of take are example, the coverage rate of its vaccine in North America be 80 ?90%, the coverage rate in Europe be about 70 ?80%, and coverage rate in Asia be only 40 ?50%.Analyze its reason place, the distribution of the streptococcus pneumoniae serotype spectrum of developed country is narrow, and the distribution of the streptococcus pneumoniae serotype spectrum of undeveloped country is broader.Basic reason that why 7 valency pneumococcal Polysaccharide Conjugate Vaccines of U.S. Wyeth cannot be applied in Asia that Here it is.According to nearly 5 years of China and south east asia clinically 0 ?the child of 5 years old, the pneumococcal report on Epidemiological of 2 years old Infants Below particularly, find that there is 13 kinds of serotypes substantially contained 80 ?90% popular bacterial strain, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F.Accordingly, Pfizer develops 13 valency pneumococcal Polysaccharide Conjugate Vaccines, and the GSK in Europe has developed 10 valency pneumococcal Polysaccharide Conjugate Vaccines.

In the serious infectious disease such as the pneumonia that pneumococcal Polysaccharide Conjugate Vaccine is caused by pneumococcal infection at preventing child, meningitis, otitis media, brought into play huge effect.But, on market, the immunogenicity variability of existing pneumococal polysaccharide protein binding vaccine is larger, this variability is except being the reason due to the structural difference of different serotypes polysaccharide, the use of different immunogenic protein carriers is also the immunogen difference main cause that causes 13 valency pneumococal polysaccharide protein conjugates.In some high-risk group, such as children's, old people or immunologic hypofunction people, the immune effect of the proteinpolysaccharide combined vaccine of reduced immunogenicity is not good, and protectiveness is restricted.Therefore, develop the pneumococal polysaccharide protein binding vaccine that immunogenicity is stronger, remain the direction that make great efforts in this field.

Summary of the invention

The object of this invention is to provide a kind of immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates, the omnipotent epi-position peptide protein carrier P30CRM197A that contains that adopts gene recombination technology production introduces omnipotent epitope peptide P30 in proteinpolysaccharide conjugate.At this GL-PP conjugate, enter after animal body, after antigen-presenting cell (APC) is engulfed digestion degraded, its omnipotent epitope peptide P30 and part pneumococal polysaccharide recurring unit, ajor histocompatibility complex ClassII combination, can effectively stimulate T cell, and then the immunogenicity of pneumococal polysaccharide in enhancing conjugate, cause the antibody concentration that is directed to pneumococcal capsular polysaccharide to increase.

The technical scheme that the present invention takes is as follows:

Strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that:

Step 1: add omnipotent epitope peptide FNNFTVSFWLRVPKVSASHLE (being called for short P30) in the A chain (being called for short CRM197A) of diphtheria toxin, diphtherotoxin variant CRM197, produce the CRM197A protein carrier (being called for short P30CRM197A) that contains P30 by gene recombinaton engineering bacteria;

Step 2: by the pneumococcal capsular polysaccharide of 13 kinds of different serotypes, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F, by covalent bond respectively with P30CRM197A protein carrier be connected to form 13 kinds of unit price pneumococcus Duo Tang ?P30CRM197A conjugate;

Step 3: 13 kinds of unit price pneumococcus Duo Tang that step 2 is obtained ?P30CRM197A conjugate be mixed to get the 13 valency pneumococal polysaccharide protein conjugates that immunogenicity strengthens.

Further, in described P30CRM197A protein carrier, contain X P30,1≤X≤3.

Further, in described P30CRM197A protein carrier, P30 be attached at CRM197A albumen N ?end or C ?end, or be connected to simultaneously N ?end and C ?end.

Further, between described P30 and CRM197A albumen, be to be connected by GSGSG aminoacid sequence.

Further, described gene recombinaton engineering bacteria is the escherichia coli that build by gene recombination method.

Further, described pneumonia capsular polysaccharide is the capsular polysaccharide obtaining by cultivating respectively the streptococcus pneumoniae of 13 different serotypes.

The specific embodiment

Illustrate specific embodiment of the invention method below, but be not only confined to following instance.

The first step, the preparation of carrier protein and pneumococal polysaccharide

For effectiveness of the present invention is described, prepared two kinds of carrier proteins, contain the protein carrier P30CRM197A of P30 and not containing the protein carrier CRM197A of P30.Wherein, CRM197A protein carrier be for the synthesis of contrast with 13 valency pneumococcus Duo Tang ?CRM197A conjugate sample.

One, the design of CRM197A protein carrier and P30CRM197A protein carrier aminoacid sequence

1, the design of CRM197A protein carrier aminoacid sequence

Diphtheria toxin, diphtherotoxin is by expressing in diphtheria corynebacterium with the phagus beta of diphtheria toxin, diphtherotoxin gene, and in antibacterial endochylema, existence form is polypeptide, 560 aminoacid, consists of, and molecular weight is 62,000 dalton.Its aminoacid sequence is as follows:

MSRKLFASILIGALLGIGAPPSAHA GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKS GTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETI KKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFET RGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Before secreting to bacterial body, polypeptide N ?25 of end guiding aminoacid sequences are cut falls, become by 535 aminoacid and formed, the single chain polypeptide that molecular weight is 58kD is secreted to bacterial body, its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDA AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRF GDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR VRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

Secreting diphtheria toxin, diphtherotoxin to bacterial body digested is A chain and B chain, is connected therebetween and form a protein molecular by a disulfide bond.These two polypeptide chains have different functions, A chain be DT molecule N ?terminal fragment, molecular weight is 21kD, 193 aminoacid, consists of.It is the toxicity funtion part of diphtheria toxin, diphtherotoxin, by eukaryotic cells slurry, by the Isocitrate dehydrogenase (NAD of diphosphonic acid three adenosine ribose (ADP ?Ribosyl) ⁺) part be transferred to elongation factor 2 (Elongation Factor ?2, EF ?2) upper, thereby suppress the protein synthesis in cell, and then cell growth inhibiting, causes cell injures and deaths.B chain be DT molecule C ?terminal fragment, molecular weight is approximately 37kD, 342 aminoacid, consists of.The function of B chain is the specific receptor on identification sensitive cells surface, and diphtheria toxin, diphtherotoxin is adsorbed on sensitive cells, helps A chain to enter in cell.

The A chain of diphtheria toxin, diphtherotoxin has good water solublity, and its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDA AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRF GDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR VRR

[3] are found in research, due to the sudden change of the toxin gene tox on phagus beta, for the not impact that copies of phage; But the toxicity of the toxin being synthesized may disappear, or reduce widely, and form diphtheria toxin mutation (Cross Reacting Material, CRM), and the serology immunogenicity of diphtheria toxin mutation is still associated with toxin.Such as, diphtheria toxin muton CRM 197, without the toxicity of diphtheria toxin, diphtherotoxin, is due to the 52nd amino acids A chain from amino acid sequence analysis, by glycine (Gly), is mutated into glutamic acid (Glu), its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRF GDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR VRR

Experimental study shows, with on other present market for the synthetic protein carrier comparison of pneumococcal conjugated vaccine, diphtheria toxin, diphtherotoxin variant CRM197 protein A chain polypeptide possesses following advantages, as its immunogenicity and diphtheria toxin, diphtherotoxin and diphtheria endotoxin is associated, molecular weight is little, water solublity is high, be easy to produce and carry out macromole synthetic reaction.The clinical use of long-term diphtheria toxoid vaccine, verified its safety and effectiveness.

The design of the P30CRM197A protein carrier aminoacid sequence that 2, contains the omnipotent epitope peptide of P30

Omnipotent epitope peptide P30 is connected on CRM197A protein carrier, constructs a kind of new protein carrier for the synthesis of proteinpolysaccharide conjugate.Omnipotent epitope peptide P30 used can be connected to N ?end or the C ?end of CRM197A protein carrier; Also two different omnipotent epitope peptides can be connected to respectively to N ?end or the C ?end of CRM197A protein carrier; Another kind of mode is that two omnipotent epitope peptides self are connected, and then is connected to CRM197A protein carrier N ?end or C ?end; Also having a kind of mode is that an omnipotent epitope peptide is connected to C ?end or N ?end, and two omnipotent epitope peptides that self connect connect the other end.

2 ?1, the design of P30CRM197A protein carrier aminoacid sequence that contains omnipotent epitope peptide P30

2 ?1 ?1, P30 ?N ?the design of end CRM197A protein carrier (be called P30 ?CRM197A) aminoacid sequence

By P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE is added to CRM197A protein carrier N ?end, form a new albumen, its aminoacid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Between the N ?end of P30 aminoacid sequence and CRM197A protein carrier, inserted GSGSG fragment and be connected, the albumen building with the method is called P30 ?CRM197A protein carrier.

2 ?1 ?2, CRM197AC ?Mo Duan ?P30 protein carrier (be called CRM197A ?P30) aminoacid sequence design by P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE is added to CRM197A protein carrier C ?end, form another kind of new albumen, its aminoacid sequence is as follows:

MGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG FNNFTVSFWLRVPKVSASHLE

Between the C ?end of P30 aminoacid sequence and CRM197A protein carrier, inserted GSGSG fragment and be connected, the albumen building with the method is called CRM197A ?P30 protein carrier.

2 ?1 ?3, P30 ?N ?end CRM197AC ?Mo Duan ?P30 protein carrier (be called P30 ?CRM197A ?P30) aminoacid sequence design by two P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE are added to respectively CRM197A protein carrier N ?end and C ?end, form a kind of new albumen, its aminoacid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG FNNFTVSFWLRVPKVSASHLE

The N of P30 aminoacid sequence and CRM197A protein carrier ?end and C ?inserted GSGSG fragment between end and connected, the albumen building with the method be called P30 ?CRM197A ?P30 protein carrier.

2 ?1 ?4, P30 ?P30 ?N ?end CRM197A protein carrier (be called P30 ?P30 ?CRM197A) design of aminoacid sequence is by connecting two P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE self, and then be added to CRM197A protein carrier N ?end, form a kind of new albumen, its aminoacid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSG FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRR

Between two P30 aminoacid sequences that self connect, and with the N ?end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called P30 ?P30 ?CRM197A protein carrier.

2 ?1 ?5, CRM197AC ?Mo Duan ?P30 ?P30 protein carrier (be called CRM197A ?P30 ?P30) design of aminoacid sequence is by connecting two P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE self, and then be added to CRM197A protein carrier C ?end, form a kind of new albumen, its aminoacid sequence is as follows:

GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG FNNFTVSFWLRVPKVSASHLEGSGSG FNNFTVSFWLRVPKVSASHLE

At two, self connect between P30 aminoacid sequence, and with the C ?end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called CRM197A ?P30 ?P30 protein carrier.

2 ?1 ?6, P30 ?P30 ?N ?end CRM197AC ?Mo Duan ?P30 protein carrier (be called P30 ?P30 ?CRM197A ?P30) design of aminoacid sequence is by two P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE self are connected, and then be added to CRM197A protein carrier N ?end; In addition, by a P30 be added to CRM197A protein carrier C ?end, form another kind of new albumen, its aminoacid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSG FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG FN NFTVSFWLRVPKVSASHLE

At two, self connect between P30 aminoacid sequence, and with the C ?end of CRM197A albumen between inserted GSGSG fragment and be connected, the albumen building with the method is called P30 ?P30 ?CRM197A ?P30 protein carrier.

2 ?1 ?7, P30 ?N ?end CRM197AC ?Mo Duan ?P30 ?P30 protein carrier (be called P30 ?CRM197A ?P30 ?P30) design of aminoacid sequence

By a P30 aminoacid sequence FNNFTVSFWLRVPKVSASHLE is connected to CRM197A protein carrier N ?end; In addition, two P30 are carried out to self and connect, and then be added to CRM197A protein carrier C ?end, form another kind of new albumen, its aminoacid sequence is as follows:

FNNFTVSFWLRVPKVSASHLEGSGSGGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRGSGSG FNNFTVSFWLRVPKVSASHLEGSGSG FN NFTVSFWLRVPKVSASHLE

At two, self connect between P30 aminoacid sequence, and with the C ?end of CRM197A protein carrier between inserted GSGSG fragment and be connected, the albumen building with the method is called P30 ?CRM197A ?P30 ?P30 protein carrier.

Two, the structure of CRM197A protein carrier and the P30CRM197A protein carrier expression plasmid that contains omnipotent epitope peptide

1, the structure of CRM197A protein carrier expression plasmid

From GenBank, obtain CRM197 albumen complete amino acid sequence PRF:224021, determine the A chain fragment of CRM197 albumen, the aminoacid of CRM197 1 ?the 193 A chain fragments that are CRM197.On this basis, the amino acid whose nucleotide sequence of this fragment is optimized, so as in escherichia expression system high efficient expression.Adopt homemade expression plasmid, with Nde I enzyme identification plasmid site CATATG, Bam HI enzyme recognition site GGATCC.Through the gene order of CRM197A is analyzed, in sequence, without Nde I and Bam HI restriction enzyme site.CRM197A protein gene composition sequence is as follows:

CATATG

GGTGCGGACGACGTTGTGGACTCCTCAAAATCGTTTGTCATGGAAAACTTCAGCTCTTAT

CATGGCACCAAACCGGGTTACGTGGACTCCATTCAGAAGGGCATCCAAAAACCGAAGTCA

GGCACCCAGGGTAACTACGATGACGATTGGAAG GAATTCTACAGCACGGACAATAAGTAT

GATGCGGCCGGCTACTCTGTTGACAACGAAAATCCGCTGAGTGGTAAAGCAGGCGGTGTG

GTTAAGGTCACCTATCCGGGTCTGACGAAAGTTCTGGCGCTGAAGGTCGATAACGCCGAA

ACCATTAAAAAGGAACTGGGCCTGTCTCTGACCGAACCGCTGATGGAACAAGTGGGTACG

GAAGAATTTATCAAACGTTTCGGCGATGGTGCATCGCGTGTCGTGCTGAGCCTGCCGTTT

GCTGAAGGCAGTTCCTCAGTGGAATACATTAACAATTGGGAACAAGCAAAAGCTCTGTCA

GTTGAACTGGAAATCAATTTCGAAACGCGTGGCAAACGCGGTCAAGATGCTATGTATGAA

TATATGGCTCAGGCGTGTGCGGGCAATCGCGTCCGTCGCTAA

GGATCC

By in the PCR product of blank plasmid and synthetic CRM197A protein gene, add respectively Nde I enzyme and Bam HI enzyme to carry out double digestion reaction; After purification, in linked system, add T4 ligase to connect, purification expression plasmid after completing, and identify with PCR identification system and restriction enzyme mapping.With BL21 (DE3) competent cell, expression plasmid is converted in cell to screening and cloning.Obtain after positive expression engineering bacteria, set up seed bank, comprise main seed and work seed, and Chu Cun Yu ?in 20 ℃ of following refrigerators.

The structure of the P30CRM197A protein carrier expression plasmid that 2, contains omnipotent epitope peptide

2 ?1, P30 ?the structure of CRM197A protein carrier expression plasmid

Adopt homemade blank expression plasmid, with Nde I enzyme identification plasmid site CATATG, Bam HI enzyme recognition site GGATCC.Through P30CRM197A protein carrier gene order is analyzed, in sequence, without Nde I and Bam HI restriction enzyme site.P30 ?the synthetic complete sequence of CRM197A gene as follows:

CATATG

TTCAATAATTTTACGGTGTCGTTTTGGCTGCGTGTCCCGAAAGTCTCTGCGAGTCATCTG

GAAGGTTCTGGTAGCGGTGGTGCGGATGACGTGGTTGATAGCTCTAAATCTTTCGTTATG

GAAAACTTCAGTTCCTATCATGGCACCAAACCGGGTTACGTCGATTCGATTCAGAAAGGC

ATCCAAAAACCGAAAAGCGGCACCCAGGGTAACTACGATGACGATTGGAAAGAATTCTAC

TCAACGGACAACAAATACGATGCGGCCGGCTACTCCGTGGACAACGAAAATCCGCTGAGC

GGTAAAGCGGGCGGTGTCGTGAAAGTTACCTATCCGGGTCTGACGAAAGTGCTGGCTCTG

AAAGTTGATAATGCGGAAACCATCAAAAAAGAACTGGGCCTGTCCCTGACCGAACCGCTG

ATGGAACAAGTGGGTACGGAAGAATTTATCAAACGTTTCGGCGACGGTGCCTCTCGCGTT

GTCCTGAGTCTGCCGTTTGCAGAAGGCTCATCGAGCGTCGAATACATTAACAATTGGGAA

CAAGCAAAAGCTCTGAGCGTGGAACTGGAAATCAACTTCGAAACGCGTGGCAAACGCGGT

CAGGATGCGATGTATGAATACATGGCGCAAGCCTGCGCAGGTAATCGTGTTCGTCGCGGATCC

By blank plasmid and synthetic P30 ?in the PCR product of CRM197A protein gene, add respectively Nde I enzyme and Bam HI enzyme to carry out double digestion reaction; After purification, in linked system, add T4 ligase to connect, purification expression plasmid after completing, and identify with PCR identification system and restriction enzyme mapping.With BL21 (DE3) competent cell, expression plasmid is converted in cell, screening and cloning, and identify with PCR identification system and restriction enzyme mapping.Obtain after positive expression engineering bacteria, set up seed bank, comprise main seed and work seed, and Chu Cun Yu ?in 20 ℃ of following refrigerators.

2 ?2, CRM197A ?P30, P30 ?CRM197A ?P30, P30 ?P30 ?CRM197A, CRM197A ?P30 ?P30, P30 ?P30 ?CRM197A ?P30 and P30 ?CRM197A ?P30 ?the structure of P30 protein expressing plasmid:

Method and upper joint " 2 ?1, P30 ?the structure of CRM197A protein expressing plasmid " identical.

Three, the preparation that contains omnipotent epitope peptide P30CRM197A protein carrier and CRM197A protein carrier

Experimental result of the present invention shows, CRM197A protein carrier is similar with the characteristic of the CRM197A protein carrier that contains omnipotent epitope peptide; Therefore, the purification process of these protein carriers is similar, and below to take the CRM197A protein carrier that contains omnipotent epitope peptide be example illustration method.

1, express the preparation that contains omnipotent epitope peptide P30CRM197A protein carrier engineering bacteria

By standard molecular biological method, the plasmid of each expressing protein carrier is proceeded to competent cell, and express calibrating.Filter out expressing quantity high, and by the clone with antiserum assay approval, set up main seed bank and work seed bank.

2, contain the fermentation that omnipotent epitope peptide P30CRM197A protein carrier is expressed engineering bacteria

From colibacillus engineering work seed bank cryogenic refrigerator, take out a work seed pipe that contains omnipotent epitope peptide P30CRM197A protein carrier, at room temperature thaw; Be transferred in the culture medium of 50 milliliters the bacterium liquid in seed pipe is aseptic, at 37 ℃, in the shaking table that to shake speed be 180rpm, be cultured to OD ₆₀₀=1.0 left and right; By in the culture medium of bacterium liquid aseptic inoculation to 1 liter, at 37 ℃, in the shaking table that to shake speed be 180rpm, be cultured to OD ₆₀₀=1.0 left and right; Inoculation seed liquor to 50 rises in 20 liters of culture medium in fermentation tank, at 37 ℃, under 240rpm condition, ferments, and works as OD ₆₀₀to 7 ?8 o'clock, add IPTG induction recombiant protein synthetic in antibacterial; After fermentation to 14 hour, stop fermentation, centrifugal, collect thalline stand-by.

The purification of the P30CRM197A protein carrier that 3, contains omnipotent epitope peptide

The protein carrier that contains different omnipotent epitope peptides due to structure is all to take CRM197A as main body, experiment shows, although added omnipotent epitope peptide, but the parameter influence to protein purification is little, only need on the technological parameter of purification CRM197A protein carrier, carry out some and modify, just can set up the purification process of other CRM197A protein carriers that contain omnipotent epitope peptide.

The 50g wet thallus of weighing rises in Centrifuge Cup in 2 ?, adds 300ml1xPBSpH7.0 buffer suspendible thalline, stirring and evenly mixing 30min on magnetic stirring apparatus; At 4 ℃, 4000rpm, centrifugal 20min, abandons supernatant, collects thalline; Repeat this step twice; Add 300ml1xPBS pH7.0 to thalline centrifuge tube, on homogenizer, carry out fragmentation; At 4 ℃, 10000rpm, centrifugal 20min; Collecting precipitation, abandons supernatant; Add 300ml1xPBS pH7.0 buffer, on magnetic stirring apparatus, stir 30min; At 4 ℃, 4000rpm, centrifugal 20min; Abandon supernatant, collect inclusion body, add the inclusion body after 900ml degeneration buffer extremely washs, at 25 ℃, the centrifugal 30min of 10000rpm, collects supernatant, abandons precipitation; By centrifuged supernatant be transferred to 6 ?in 8KD bag filter, sealing bag filter; Put bag filter in 10 liters of renaturation buffers 1, under room temperature, on magnetic stirring apparatus, stir dialysed overnight; Proceed to bag filter in 10 liters of renaturation buffers 2 next day, stirring at room dialyse 8 ?10 hours; Bag filter is proceeded in 10 liters of renaturation buffers 3 to stirring at room dialysed overnight; Proceed to bag filter in 10 liters of renaturation buffers 4 next day, stirring at room dialyse 8 ?10 hours; Bag filter is proceeded in 10 liters of renaturation buffers 5 to stirring at room dialysed overnight; Next day by bag filter proceed to 2 liters deposit buffer in, stirring at room dialyse 8 ?10 hours; Change deposit buffer secondary, room temperature dialysed overnight; Get 1ml dialysis solution, the centrifugal 10min of room temperature 12000rpm, collects supernatant, surveys protein concentration; DEAE glue post by protein solution loading to pre-balance, with Gradient elution, and collects destination protein peak; Then loading to Phenyl drainage column is further purified, and collects eluting peak; Last loading SP glue post, collects eluting peak; The purification destination protein collect obtaining is gone in bag filter, in the sodium chloride of 0.15M, dialyses, after completing, be transferred at 4 ℃, store stand-by.

Four, the preparation of bacterial capsule polysaccharide

The present invention is to pneumococcal 13 kinds of serotype pneumococcal capsular polysaccharides, comprise 1,3,4,5,6A, 6B, 7F, 9V, 14,18C, 19A, 19F and 23F, carry out purification, for use in synthesizing of polysaccharide conjugate, its quality reaches the polysaccharide quality standard of the synthetic GL-PP combined vaccine of WHO.

1, the foundation of streptococcus pneumoniae seed bank

13 serotype streptococcus pneumoniae buying from ATCC, 9163), 3 (article No.s: 10813), 4 (article No.: BAA ?334), 5 (article No.: BAA ?341), 6A (article No.: BAA ?659), 6B (article No.: 700675), 7F (article No.: 10351), 9V (article No.: 700671), 14 (article No.s: 6314), 18C (article No.: 10356), 19A (article No.: 700673), 19F (article No.: 700905) and 23F (article No.: 700669) (the article No.: that comprises 1.Take out the strain (primordial seed is criticized) that ATCC buys, add the streptococcus pneumoniae AHC fluid medium of 0.5ml that strain is mixed, get 0.25ml bacterium liquid in 5% Sanguis caprae seu ovis AHC culture fluid.Postvaccinal 5% Sanguis caprae seu ovis AHC culture fluid pipe is placed on the cultivation shaking table of 36 ℃ ± 1 ℃, shakes fast 120rpm, cultivate 12 ?20 hours.Treat OD ₆₀₀to 1.0 o'clock, with inoculating loop, 5% Sanguis caprae seu ovis AHC culture fluid is seeded on AHC agar culture plate, be placed in the incubator of 36 ℃ ± 1 ℃, cultivate 12 ?20 hours.With inoculating loop, 1 on AHC agar plate, in the AHC culture fluid to several colony inoculations in 10ml, is placed in to 36 ℃ ± 1 ℃, cultivates 12 hours cultivating on shaking table, shake fast 150 ?200rpm.Antibacterial OD in culture fluid ₆₀₀grow to 1.0, take out 5ml antibacterial AHC culture fluid and be inoculated in the fresh AHC culture fluid of 200ml, be placed on the cultivation shaking table of 36 ℃ ± 1 ℃ and cultivate about 12 hours, shake fast 150 ?200rpm.OD ₆₀₀to 1.0 o'clock, inoculum is sub-packed in 200 small test tubes to centrifugal (4000rpm with 1ml, 10min), remove supernatant culture fluid, then add the fresh AHC culture fluid of 0.5ml and the germfree defatted milk of 0.5ml, mix quick freezing on ethanol dry ice.Vacuum freeze-drying, numbering, is stored in 4 ℃ of refrigerators as main seed lot.Take out main seed lot and set up, according to main seed method for building up, inoculum is inoculated in the fresh AHC culture fluid of another 200ml, be placed on the cultivation shaking table of 36 ℃ ± 1 ℃ and cultivate 12 hours, shake fast 150 ?200rpm.Treat OD ₆₀₀to 1.0 o'clock, inoculum is sub-packed in 200 small test tubes with 1ml, centrifugal (4000rpm, 10min), removes supernatant culture fluid, then adds the fresh AHC culture fluid of 0.6ml and 40% glycerite of 0.4ml, mixes.Quick-freezing on dry ice, as work seed Bao Cun Yu ?in 70 ℃ of cryogenic refrigerators.

2, pneumococcal fermentation

From seed bank, take out lyophilizing work seed pipe, add 1mlAHC enrichment culture liquid to dissolve lyophilised bacteria.The bacterium liquid of dissolving is inoculated in 5ml AHC enrichment culture liquid test tube, at CO ₂standing overnight incubation in incubator.When observation has bacterial growth, bacterium liquid is inoculated in the AHC enrichment culture liquid triangular flask of 100ml.Put culture bottle in shaking table, at 36 ℃, 200rpm/min is cultured to OD ₆₀₀be 1.0.By 100ml inoculum be inoculated in respectively 2 be equipped with 1 ?in the AHC enrichment culture liquid bottle that rises.Put culture bottle in shaking table, at 36 ℃, 200rpm/min is cultured to OD ₆₀₀be 1.0.The aseptic filtration AHC enrichment culture liquid of 35 liters is injected to 50 liters of fermentation tanks.Inoculate bacterium liquid that 2 liters of OD are 1 to fermentation tank.When bacterial growth arrives plateau, sterilization, results culture supernatant.

3, the purification of capsular polysaccharide

Use deep layer membrane filtration, further remove remaining antibacterial and fragment.By aseptic 100Kd ten times of ultrafiltration and concentration of ultrafilter membrane (about 600ml) for supernatant culture fluid.With 6 liters of 25mM sodium acetate solution, carry out ultrafiltration cleaning.Adding HB storage liquid to make ultimate density is 1% (w/v), mixes, and solution is positioned over to freezer and spends the night.Centrifugal, 4000rpm, 1 hour, collect polysaccharide/HB precipitation, abandon centrifugal liquid.Add the sodium acetate+1%HB of 25mM to polysaccharide/HB precipitation vessel, stirring suspension precipitation, centrifugal, 4000rpm, 1 hour, collect polysaccharide/HB precipitation, abandon centrifugal liquid.Repeat 3 times this step.0.25M sodium chloride solution dissolution precipitation with 600ml.Centrifugal, 4000rpm, 1 hour, abandons insoluble contaminant nucleic acid.Add 10% liquor kalii iodide to enter in polysaccharide+HB mixed solution, mix, potassium iodide ultimate density is 0.5%, solution is placed in to freezer and spends the night.Use deep bed filter filtering solution, remove the HB/I precipitation in solution, and clean the precipitation on deep bed filter with 0.25M sodium chloride/0.5% liquor kalii iodide, collect filtrate, discard precipitation.Rough polysaccharide solution is passed into active carbon deep bed filter and carry out circulating filtration 30 minutes (4% active carbon/0.5mg/ml polysaccharide crude solution).Add sodium phosphate buffer pH6.8, ultimate density 25mM joins in polysaccharide solution.Above solution is crossed to HA post (50 ?100ml), circulate 30 minutes.With identical phosphate solution wash post 4 ?5 column volumes.With 5 times of the concentrated polysaccharide solutions of 30Kd membrane ultrafiltration, use without heat source water ultrafiltration and clean polysaccharide solution.With 0.22 μ m membrane filtration, lyophilizing.

Second step: 13 kinds of serotype unit price pneumococcus Duo Tang ?the preparation of P30CRM197A conjugate

In the chemical constitution of different streptococcus pneumoniae serotype capsular polysaccharides, contain different groups, need to adopt different synthetic methods by polysaccharide covalent key be connected to protein carrier and form conjugate, and output and the immunogenicity of the synthetic conjugate of distinct methods are different.The present invention is according to experimental result, adopt three kinds of synthetic methods, reduce amine method, CDAP method (1 ?(3 ?dimethylaminopropyl) ?3 ?ethyl-carbodiimide hydrochloride method) and ADH method (adipyl dihydrazide method), synthesize specific GL-PP conjugate.

One, 13 kinds of streptococcus pneumoniae serotype Jia film Duo Tang ?P30 ?CRM197A conjugate synthetic

1, streptococcus pneumoniae serotype 1 Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take the Pn1 degradation of polysaccharide of 5mg in reaction bulb, the 1mol/L NaCl that measures 0.5ml adds in reaction bulb; Magnetic agitation is dissolved polysaccharide completely.Record the initial pH of polysaccharide solution, measure respectively appropriate CDAP solution, add in reaction bulb.Stirring reaction 1.5min under room temperature, measures the pH of solution during 30s.After 1.5min, with the pH to 9.5 of 0.2mol/L NaOH regulator solution, stirring reaction 3min under room temperature (maintaining pH 9.5 with 0.2mol/L NaOH).After 3min immediately to add in reaction bulb 5mg P30 ?CRM197A albumen, at room temperature (25 ℃) stirring reaction 1h.Measure 37.5 μ l2mol/L lysines and add in reaction bulb, with 0.1N HCl regulator solution pH to 9.0.Stirring reaction 30min under room temperature, transfers to reaction at 4 ℃ by reaction bulb and spends the night.Reactant mixture is transferred in bag filter (MWCO6 ?8000), at 4 ℃, to 0.85%NaCl solution dialysis 3 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

2, streptococcus pneumoniae serotype 3 Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take the Pn3 degradation of polysaccharide of 20mg in reaction bulb, the 0.15M NaCl that measures 2ml adds in reaction bulb; Magnetic agitation is dissolved polysaccharide completely.Measure appropriate CDAP solution, add in reaction bulb.Stirring reaction 1.5min under room temperature, measures the pH of solution during 30s.After 1.5min, with the pH to 9.5 of 0.2mol/L NaOH regulator solution, stirring reaction 3min under room temperature (maintaining pH 9.5 with 0.2mol/L NaOH).Add ultimate density be the ADH of 0.8M to reaction bulb, stirring and evenly mixing, reacts under room temperature 2 hours.Polysaccharide after derivation is gone in the bag filter of 10KD 0.15M NaCl solution is dialysed, change liquid three times.Loading G ?50 posts, with 0.15M NaCl eluting, collect void volume peak.Then be transferred in bag filter, to water dialysis, change liquid three times.Take the Pn3 polysaccharide after 5mg derivation, be dissolved in the 0.15M NaCl solution of 0.5ml, add 5mg P30 ?CRM197A albumen, after stirring and evenly mixing, add the EDC of 30mM, at room temperature react 4 hours, transfer at 4 ℃ reaction and spend the night.Reactant mixture is transferred in bag filter (MWCO6 ?8000), at 4 ℃, to 0.85%NaCl solution dialysis 3 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

3, streptococcus pneumoniae serotype 4 Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 5mg activated polysaccharide to reaction bulb, measure the 0.5M sodium phosphate buffer of 100 μ l, add in reaction bulb, measure 5mg P30 ?CRM197A albumen to reaction bulb, magnetic agitation is dissolved polysaccharide completely; Measure 0.5ml pure water and add in reaction bulb, magnetic agitation mixes; The sodium cyanoborohydride that takes 5.0mg, adds in reaction bulb.The dry bath that reaction system is placed in to 30 ℃ reacts 12h.After reaction finishes, the 0.15M sodium chloride solution that measures 1.5ml adds in reaction bulb.The sodium borohydride that takes 2.5mg, adds in reaction bulb.Reaction system is placed at 22 ℃ reacts 5h; Reactant mixture is transferred to bag filter (MWCO12 ?14Kd), at 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, the liquid measure of dialysing 6L at every turn; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

4, streptococcus pneumoniae serotype 5 Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 5.0mg activated polysaccharide, be added in reaction bulb, to the 0.5M sodium phosphate buffer that adds 100 μ l in reaction bulb; Take 4.0mg P30 ?CRM197A albumen, add in reaction bulb, measure 0.5ml pure water and add in reaction bulb, magnetic agitation makes reactants dissolved, the pH of assaying reaction system; Take 5.0mg sodium cyanoborohydride, add in reaction bulb; Reaction system is placed under room temperature and is reacted 48 hours; Take the sodium borohydride of 2.5mg, be dissolved in 10 μ l pure water, with liquid-transfering gun, mix after dissolving completely, add in reaction bulb; Reaction system is placed at 23 ℃ to stirring reaction 5 hours; Reactant mixture is transferred in bag filter (MWCO6 ?8KD), at 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, within every 5 hours, changes liquid once; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

5, streptococcus pneumoniae serotype 6A Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 6.0mg activation Pn6A polysaccharide, be dissolved in 1mL purified water, be stirred to and dissolve the rear original ph of surveying completely; With 0.1M NaOH, regulate reacting liquid pH value to 7.0; To add in reaction system 4mg P30 ?CRM197A albumen, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 18 hours; Reaction finishes rear sampling censorship; Take the Sodium borohydride of 2.7mg, add in above-mentioned reaction bulb, room temperature reaction 5 hours; Reaction finishes rear sampling censorship; Reactant mixture is transferred to bag filter, at 4 ℃, to 0.15M sodium chloride solution dialysis 5 times, 6L/ time.After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

6, streptococcus pneumoniae serotype 6B Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

The Pn6B that takes 5.0mg is dissolved in 1mL purified water, is stirred to and dissolves the rear original ph of surveying completely; With 0.1M NaOH, regulate reacting liquid pH value to 7.0; To add in reaction system 2.5mg P30 ?CRM197A albumen, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 20 hours; Take the Sodium borohydride of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter, at 4 ℃, to 0.15M NaCl solution dialysis 5 times, 6L/ time; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

7, streptococcus pneumoniae serotype 7F Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

The Pn7F polysaccharide that takes 10.0mg, is dissolved in 1mL purified water, is stirred to completely and dissolves; Drip respectively 0.1M NaOH solution to polysaccharide solution, regulate pH to 7.0; To add in reaction system 3.5mg P30 ?CRM197A albumen, stirring and evenly mixing; Take the sodium cyanoborohydride of 5.0mg, add in above-mentioned reaction bulb, room temperature reaction 20 hours; In reaction bulb, add 990 μ l pure water, stirring and evenly mixing; Take the Sodium borohydride of 2.5mg, add in above-mentioned reaction bulb, room temperature reaction 6 hours; Reactant mixture is transferred to bag filter (MWCO6000 ?8000), at 4 ℃, to 5mM succinate/0.9% sodium chloride buffer dialysis 5 times, 6L/ time; After dialysis finishes, by reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect conjugate peak, sampling censorship.

8, streptococcus pneumoniae serotype 9V Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take the Pn9V activated polysaccharide of 10mg, the sodium phosphate buffer, the 125 μ l pure water that measure 125 μ l add in reaction bulb, and magnetic agitation is dissolved polysaccharide completely; Measure 15mg P30 ?CRM197A albumen to adding in reaction bulb, stirring and dissolving is complete; Take the NaBH of 10mg ₃(CN), add in reaction bulb; Reaction system is placed at 22 ℃ and is reacted 48 hours; Take the NaBH of 2.5mg ₄, add in reaction bulb, react 5 hours at 22 ℃; By reaction mixture 10000rpm, after centrifugal 10min, get supernatant, adopt AKTA system, Sepharose CL ?polysaccharide conjugate after the dialysis of 4B gel column purification, and collect in conjunction with peak.

9, streptococcus pneumoniae serotype 14 Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take the Pn14 activated polysaccharide of 5mg, measure 1ml3.9mg P30 ?CRM197A albumen, add in reaction bulb, magnetic agitation is dissolved polysaccharide completely; Add after weak reductant sodium cyanoborohydride5mg, at 22 ℃, react 48 hours; Add strong reductant sodium borohydride2.5mg, under room temperature, react 4 hours; Reaction mixture is transferred in bag filter (MWCO12 ?14KD), with the dialysis solution rinse reaction bulb of 2ml; At 4 ℃, to 0.15M sodium chloride solution dialysis 3 times, 6L/ time, within every 5 hours, change liquid once.After dialysis finishes, collect dialysis solution, 10000rpm, gets supernatant after centrifugal 10min, adopt Sepharose CL ?the polysaccharide conjugate of 4B gel column purification after dialysing, and collect conjugate peak.

10, streptococcus pneumoniae serotype 18C Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take the Pn18C degradation of polysaccharide of 5mg, with 1mL1M sodium chloride solution, dissolve; After dissolving completely, survey its original ph; Add appropriate CDAP solution, under room temperature, stir 1.5min, add the pH to 9.0 of 0.2M NaOH solution regulator solution.Afterwards in room temperature reaction 3min; Add 10mg P30 ?CRM197A albumen, at 25 ℃, react 45min; After reaction finishes, add 37.5 μ l2M lysine solutions; At 25 ℃, reacting 30min is placed on 4 ℃ of reactions and spends the night; Reaction mixture is gone in bag filter (MWCO6000 ?8000), at 4 ℃, to 0.85% sodium chloride solution dialysis, change liquid 3 times.6L/ time, within every 5 hours, change liquid once.After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL ?4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

11, streptococcus pneumoniae serotype 19A Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 10.0mg oxidation Pn19A polysaccharide, be dissolved in the buffer of 0.5mL, put bar magnet in reaction bulb, be stirred to polysaccharide and dissolve completely; Add 10mg P30 ?CRM197A albumen; Stirring and evenly mixing, takes the sodium cyanoborohydride of 5.0mg, is added in reaction bulb, at room temperature reacts 20 hours; The sodium borohydride that takes 2.5mg, adds in reaction bulb, at room temperature reacts 5 hours; Reaction mixture is gone in bag filter (MWCO6000 ?8000), at 4 ℃, to 0.85% sodium chloride solution dialysis, change liquid 3 times.6L/ time, within every 5 hours, change liquid once; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL ?4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

12, streptococcus pneumoniae serotype 19F Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 5.2mg oxidation Pn19F polysaccharide, be dissolved in 1ml pure water, put bar magnet in reaction bulb, on magnetic stirring apparatus, under room temperature, be stirred to polysaccharide and dissolve completely; Add 3.0mg P30 ?CRM197A albumen; After stirring and evenly mixing, take the sodium cyanoborohydride of 4.9mg, add and put in reaction bulb, on magnetic stirring apparatus, keep stirring always; At 18 ℃ of room temperatures, react 24 hours; The sodium borohydride that takes 2.5mg, is added to reaction bulb; At 18 ℃ of calorstats, react 5 hours; Reactant mixture is transferred to bag filter (MWCO12 ?14000), and at 4 ℃, to buffer dialysis 5 times, the liquid measure of at every turn dialysing 6L, changes liquid once in every 5 hours; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL ?4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

13, streptococcus pneumoniae serotype 23F Jia film Duo Tang ?P30 ?CRM197A protein conjugates synthetic

Take 4.9mg oxidation Pn23F polysaccharide, be dissolved in 1ml pure water, put bar magnet in reaction bulb, on magnetic stirring apparatus, under room temperature, be stirred to polysaccharide and dissolve completely; Add 5.0mg P30 ?CRM197A albumen; The sodium cyanoborohydride that takes 5.1mg, adds and puts in reaction bulb, keeps stirring on magnetic stirring apparatus always; At 18 ℃ of calorstats, react 17 hours; The sodium borohydride that takes 2.5mg, is added to reaction bulb; At 18 ℃ of calorstats, react 5 hours; Reactant mixture is transferred to bag filter (MWCO12 ?14000), at 4 ℃, to the dialysis of 0.15M sodium chloride solution, the liquid measure of dialysing 6L at every turn.Within every 5 hours, change liquid once, totally 5 times; After dialysis finishes, collect dialysis solution, the centrifugal 10min of 10000rpm, gets supernatant, employing CL ?4B gel-purified this in conjunction with polysaccharide, and collect conjugate peak; Detect albumen and polyoses content in conjugate solution.

Two, 13 kinds of pneumococcal capsular polysaccharide P30CRM197A conjugates of other P30CRM197A protein carrier that contains omnipotent epitope peptide is synthetic

The present invention design and produce totally 7 kinds of P30 ?CRM197A protein carrier carry out synthetic GL-PP conjugate, structural similarity due to protein, when synthetic for specific streptococcus pneumoniae serotype polysaccharide, the method adopting is a kind of in reduction amine method, ADH method or CDAP method.How sugared the synthetic method of 13 kinds of streptococcus pneumoniae ?P30 ?CRM197A synthetics in prosthomere example be similar, at this, do not enumerate concrete grammar.

The 3rd step: preparation and the immunogenic assessment of 13 valency pneumococal polysaccharide P30CRM197A conjugates

By the pneumococal polysaccharide protein conjugates preparing, prepare corresponding vaccine, white mice is carried out to inoculation, blood sampling, detects polysaccharide antibody concentration and opsono-cytophagic test in serum by ELISA method, assesses the immunogenicity of different conjugates.

One, 13 valency pneumococcus Duo Tang ?preparation and the immunogenic assessment of P30CRM197A conjugate

For assessing the immunogenicity of the synthetic pneumococcal capsular polysaccharide P30CRM197A conjugate of P30CRM197A protein carrier, be better than the synthetic pneumococcal capsular polysaccharide CRM197A conjugate of CRM197A protein carrier that does not contain omnipotent epitope peptide, the present invention synthesized 13 valency pneumococcus Duo Tang ?CRM197A in contrast, carry out the assessment of immunogenicity reinforced effects.

1,13 valency pneumococcus Duo Tang ?P30 ?CRM197A combined vaccine and 13 valency pneumococcus Duo Tang ?the preparation of CRM197A combined vaccine

With the Labscale ultrafiltration system of Millipore respectively by 1,3,4,5,6A, 7F, 9V, 14,18C, 19A, it is 40 μ g/ml that 19F and 23F streptococcus pneumoniae serotype capsular polysaccharide protein conjugates solution are concentrated into polysaccharide concentration; 6B serotype conjugate solution concentration is concentrated into polysaccharide concentration and is approximately 80 μ g/ml; According to following form, calculate and add the monovalent serum type conjugate solution of respective volume to office preparation bottle.

In connection with 0.22 μ m film aseptic filtration for thing mixed liquor; Add aseptic aluminum phosphate colloid, final aluminium ion concentration is 125mg/ml; With buffer, be settled to final volume; Fill, 0.5ml/ bottle.

The preparation of the 13 valency pneumococal polysaccharide P30CRM197A combined vaccines that 2, contain other P30CRM197A protein carrier

Joint in reference " 13 valency pneumococcus Duo Tang ?P30 ?preparation and the immunogenic assessment of CRM197A conjugate " method, the present invention has prepared following 6 kind of 13 valency pneumococal polysaccharide P30CRM197A in conjunction with corresponding vaccine, and in order to immunogenicity assessment experiment, comprise 13Pn ?CRM197A ?P30,13Pn ?P30 ?CRM197A ?P30,13Pn ?P30 ?P30 ?CRM197A, 13Pn ?CRM197A ?P30 ?P30,13Pn ?P30 ?P30 ?CRM197 ?P30 and 13Pn ?P30 ?CRM197A ?P30 ?P30.

3, injection white mice inoculation and blood sampling

Get 5 ?the CM57 of 6 weeks be 70 of mices, 13 valency pneumococcus Duo Tang prepared by every injected in mice ?P30 ?CRM197A protein binding vaccine, injection capacity be 0.1ml/ prop up mice/time.Inoculation mice is divided into three groups, 13 valency pneumococcus Duo Tang of one group of injection the present invention preparation ?P30 ?CRM197A protein vaccine, another group inoculation 13 valency pneumococcus Duo Tang ?CRM197A combined vaccine in contrast, the 3rd group is polysaccharide contrast, and concrete vaccinate and collection immune serum flow sheet are as follows:

Gather mouse blood to centrifuge tube, at room temperature standing 2 hours, under 10000rpm condition centrifugal 10 minutes, with liquid-transfering gun is careful, draw centrifugal supernatant serum, be stored in 4 ℃ of refrigerators, to be checked.

4, ELISA method detects polysaccharide antibody concentration in mice serum

Prepare respectively 13 kinds of different serotypes pneumococal polysaccharide storing solution 1mg/ml (in 1 * PBS solution), be stored in 4 ℃ of refrigerators.Dilute serotype pneumococal polysaccharide to be checked storage liquid to coated buffer 2 ?4 μ g/ml, add the coated solution of 100 μ l and be coated with elisa plate in each hole, at room temperature overnight incubation.With washing plate buffer, wash 4 times, add 100 μ l sealing buffer, at room temperature hatch 2 hours, with washing plate buffer, wash 4 times, can at 4 ℃, preserve one week.

The corresponding test serum 1:10 of injected in mice combined vaccine and control sample acquisition is diluted to working prototype serum, dilution suitable multiple, joins in elisa plate the first round cumulative volume 200 μ l, from first row, start to carry out two times of serial dilutions downwards, at room temperature hatch 2 hours.With washing plate buffer, wash 4 times, add 100 μ l alkali phosphatase enzyme mark sheep anti-mouse antibodies (1:2000 dilution), at room temperature hatch 4 hours.With washing plate buffer, wash 4 times, add 100 μ l phosphorus acid ?4 ?nitro phenyl ester disodium salt substrate solutions, at 405nm, read dish.

Specificity streptococcus pneumoniae serotype polysaccharide antibody concentration results in mice serum is as following table:

Result shows, pneumococcal Polysaccharide Conjugate Vaccine of the present invention, take P30 ?CRM197A as the synthetic 13 valency pneumococcus Jia film Duo Tang of protein carrier ?P30 ?the immunogenicity of CRM197A conjugate be better than 13 valency pneumococcal capsular polysaccharide CRM197A conjugates.The IgG antibody concentration of injecting anti-specificity pneumococal polysaccharide in the mice serum after three pins is significantly higher than IgG antibody concentration in the serum of injecting a pin and two pins; Inject each serotype antibody concentration after three pins higher than injection the more than 4 times of one pin, meet the standard that WHO improves for GL-PP combined vaccine concentration.With 13 valency pneumococcus Jia film Duo Tang ?the comparison of CRM197A conjugate, 13 valency pneumococcus Jia film Duo Tang ?P30 ?the concentration of each serotype antibody of CRM197A conjugate, after injection three pins, the polysaccharide specific antibody concentration in mice serum is higher.

5, opsono-cytophagic test (Opsonophagocytic Assay, OPA)

Opsono-cytophagic test be evaluate 13 valency pneumococcus Duo Tang ?P30 ?CRM197A combined vaccine fungicidal effectiveness method, according to UAB ?" the many types of opsonophagocytosis bactericidal assay of the streptococcus pneumoniae capsular polysaccharide specific antibody method " of MOPA the mouse immune serum obtaining is tested.OPA concentration results is as following table:

Result of the test is visible, inject 13 valency pneumococcus Duo Tang after three pins ?P30 ?the merging mouse immune serum of CRM197A conjugate group and 13 valency pneumococcus Duo Tang ?the merging mouse immune serum comparison of CRM197A conjugate group, the OPA concentration of its antibody is significantly improved.

Two, other 13 valency pneumococcus Duo Tang ?preparation and the immunogenic assessment of P30CRM197A conjugate

Similar with the method for upper joint " 13 valency pneumococcus Duo Tang ?P30 ?preparation and the immunogenic assessment of CRM197A conjugate ", the antipolysaccharide antibody IgG concentration results of the how sugared ?P30CRM197A of the 13 valency streptococcus pneumoniae conjugate that other protein carrier that contains P30 is synthetic is as following table, and this table is just listed the polysaccharide IgG antibody concentration after inoculation three pins.

Result from above table, all immunogenicities that contain 13 synthetic valency pneumococal polysaccharide conjugates of the omnipotent epi-position PEPC of P30 RM197A protein carrier, do not contain 13 synthetic valency pneumococal polysaccharide conjugate comparisons of the omnipotent epi-position PEPC of P30 RM197A protein carrier, have obvious enhancing.The immunogenicity of the 13 valency pneumococal polysaccharide P30CRM197 conjugates that the CRM197A protein carrier that contains two omnipotent epitope peptides of P30 synthesizes will be higher than only containing 13 synthetic valency pneumococal polysaccharide CRM197A conjugates of the omnipotent epi-position PEPC of a P30 RM197A protein carrier.When the omnipotent epitope peptide quantity of the P30 in CRM197A protein carrier is increased to three, and the immunogenicity of the synthetic 13 valency pneumococal polysaccharide P30CRM197A conjugates of the protein carrier that contains two omnipotent epitope peptides of P30 is relatively without significant change.The omnipotent epitope peptide of P30 in CRM197A protein carrier is added to N ?end and there is no difference with the 13 valency pneumococal polysaccharide P30CRM197A conjugate immunogenicities that add to C ?end.

List of references

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Claims

1. strengthen the immunogenic method of 13 valency pneumococal polysaccharide protein conjugates, it is characterized in that:

2. the immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates according to claim 1, is characterized in that: in described P30CRM197A, contain X P30,1≤X≤3.

3. the immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates according to claim 1, it is characterized in that: in described P30CRM197A protein carrier, P30 be attached at CRM197A albumen N ?end or C ?end, or be connected to simultaneously N ?end and C ?end.

4. according to the immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates described in claim 1,2 or 3, it is characterized in that: in described P30CRM197A protein carrier, between P30 and CRM197A albumen, by GSGSG aminoacid sequence, be connected.

5. the immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates according to claim 1, is characterized in that: described gene recombinaton engineering bacteria is the escherichia coli that build by gene recombination method.

6. the immunogenic method of enhancing 13 valency pneumococal polysaccharide protein conjugates according to claim 1, is characterized in that: described pneumonia capsular polysaccharide is the capsular saccharides obtaining by cultivating respectively the streptococcus pneumoniae of 13 different serotypes.