WO2019170068A1 - 一种肺炎链球菌疫苗及其制备方法 - Google Patents

一种肺炎链球菌疫苗及其制备方法 Download PDF

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WO2019170068A1
WO2019170068A1 PCT/CN2019/076919 CN2019076919W WO2019170068A1 WO 2019170068 A1 WO2019170068 A1 WO 2019170068A1 CN 2019076919 W CN2019076919 W CN 2019076919W WO 2019170068 A1 WO2019170068 A1 WO 2019170068A1
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group
pneumoniae
gene
streptococcus pneumoniae
capsular polysaccharide
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PCT/CN2019/076919
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French (fr)
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吴克
陈煜�
杜林森
梁锦
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武汉博沃生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus

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  • the invention relates to a pneumococcal vaccine and a preparation method thereof, and belongs to the field of biopharmaceutical.
  • Streptococcus pneumoniae which resides in the nasopharyngeal cavity of normal people, is the main pathogen of bacterial lobar pneumonia, meningitis, otitis media, pneumonia, and bronchitis. Diseases caused by S. pneumoniae have been a serious public health problem worldwide, with high morbidity and mortality worldwide, especially for children under 2 years of age and older people.
  • the currently marketed S. pneumonia capsular saccharide vaccine and capsular glycoprotein conjugate vaccine are designed based on S. pneumonia capsular saccharide and cover the most common serotypes that cause pneumococcal disease.
  • the Streptococcus pneumoniae capsular saccharide is a Thymus independent antigen (TI-Ag).
  • the antibody response mainly depends on the linear epitope of its repeat unit composition, and directly with B lymphocytes without T lymphocyte help.
  • IgM receptor cross-linking on the cell surface the induced antibodies are mainly IgM and IgG2, lacking good complement activation ability, antibody levels can not be maintained for a long enough time, and can not induce immune memory, can not be produced in children under 2 years old Immune protection.
  • the complex structure of capsular sugars results in different immunogenicity of each serotype and does not produce an effective immune response.
  • Streptococcus pneumoniae conjugate vaccines include a large number of serotypes, and the specific structures of the types used for binding differ, resulting in different binding methods for each type.
  • the modification of the capsular saccharide and the binding to the carrier protein should be carried out under the premise that the capsular saccharide-specific group is not lost, the antigenicity and the immunogenicity are not affected, and at the same time, in order to avoid excessive cross-linking and conjugate of the sugar chain For sterilization filtration requirements, there should be some control over the size of the capsular sugar and conjugate molecules.
  • the seven-valent vaccine was approved for use in the United States in February 2000. Since there are many types of Streptococcus pneumoniae, it is necessary to combine protein components in the production process of the conjugate vaccine, and since the protein component can cause local reactions, it is difficult to produce a conjugate vaccine containing more than 12 types.
  • the antibody concentration of the conjugate vaccine after the initial immunization can only last for several months, and then it will drop to the pre-immune level; and the entire process of combining the vaccine requires the addition of various chemical reagents to participate in the reaction, and the capsular glycoprotein conjugate vaccine.
  • the low serotype coverage and increased non-vaccine serotypes of S. pneumoniae infectious diseases have led to more researchers focusing on the development of other S. pneumonia vaccines.
  • Streptococcus pneumoniae Due to its serotype, Streptococcus pneumoniae has poor antigenic structure of the antigen itself, and it is difficult to coexist with other vaccines.
  • the existing S. pneumoniae capsular glycoprotein conjugate vaccine is treated with diphtheria or tetanus toxoid.
  • the protein carrier, the main component of this vaccine is a serotype-specific capsular saccharide, which is ineffective against other serotypes of S. pneumoniae infection not included in the vaccine, ie lack of cross-immunoprotective effect; and will be routinely immunized with children.
  • the diphtheria and tetanus vaccines used in the vaccination interfere with the existing immune effects. Therefore, it is imperative to study a vaccine that can cross the serotype S. pneumoniae protein vaccine.
  • the whole serotype immunization direction that the technicians mainly try is the direction of the protein vaccine, that is, the stable protein which can stimulate the immune response in the pneumococcal strain is extracted as an antigen for the immune reaction, but the protein is not only greatly reduced in vitro, but also causes strong The immune side effects are too strong for the immune process in infants with poor tolerance.
  • the physicochemical properties of protein vaccines and Streptococcus pneumoniae cells are very different. Although some scientific research institutions have indicated that the successful development of the pneumococcal protein vaccine, there are no varieties that have been approved for marketing, indicating that this direction requires a certain clinical The process of argumentation.
  • the main pathogenic factor of Streptococcus pneumoniae is the capsule.
  • the main chemical component of the capsule is capsular polysaccharide.
  • capsular polysaccharide involves a series of complex biochemical processes, which block any step in the expression of capsular polysaccharide, or interfere with key enzymes and key reactants in the expression process to affect the expression or synthesis of related capsular polysaccharides. The steps will affect the final formation of the capsular polysaccharide. Therefore, the applicant has designed a vaccine for producing a capsule-free S. pneumoniae as an antigen and a preparation method of the vaccine by focusing on scientific research.
  • the S. pneumoniae vaccine comprises at least one serotype of Streptococcus pneumoniae, which is a capsule-free membrane after knocking or modification affects the capsular polysaccharide synthesis pathway and/or affects the capsular polysaccharide expression-related gene.
  • Knocking or modifying any one or more of the related genes in the capsular polysaccharide expression or synthesis pathway may affect the expression or synthesis pathway of the entire capsular polysaccharide, ie, affect the formation of the final capsular polysaccharide.
  • any gene which can affect the formation of capsular polysaccharide can be used as a knock-out or modification object in the present invention.
  • gene knockout or modification refers to a technique of inactivating or deleting a body-specific gene by a certain route. Any biological method in which gene inactivation or deletion can be performed can be used as the "knockout or modification" method in the present invention.
  • the S. pneumoniae is selected from the group consisting of one or more of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41, 41F, 42, 43, 44, 45, 46, 47A, 47
  • Streptococcus pneumoniae is currently reported to have a total of 91 serotypes. Although only a few serotypes are pathogenic, each serotype has a basic capsular structure and can be used as an antigen modification of the non-capsulated pneumococcal vaccine of the present invention. source.
  • the S. pneumoniae vaccine is a single serotype S. pneumoniae vaccine. Since the non-capsulated pneumococcal vaccine has the characteristics of breaking through the serotype restriction, the single serotype modification can exert a good immune effect.
  • Each pathogenic serotype is preferably modified, and the S. pneumoniae is selected from one of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F.
  • the gene is selected from one or a combination of the following genes: cps gene, galU, CcpA, and/or PGM.
  • the cps gene is selected from one or a combination of the following genes: cpsA, cpsB, cpsC, cpsD, CpsE, CpsF, CpsG and/or CpsI.
  • CcpA may bind to the promoter region of the S. pneumoniae capsular polysaccharide (CPS) locus, and CcpA (catabolite control protein A, CcpA) is a transcriptional regulator involved in glucose metabolism, regulating various genes of S. pneumoniae. Expression; glucose phosphate mutase (PGM), galU (uridine triphosphate-glucose-1-phosphate uridine acyltransferase), the above gene loci can be knocked out individually or in combination to prepare a capsule-free or capsule-deficient Streptococcus pneumoniae.
  • PGM glucose phosphate mutase
  • galU uridine triphosphate-glucose-1-phosphate uridine acyltransferase
  • the present application knocks out or modifies a gene which has been confirmed to have capsular polysaccharide synthesis as a guest of genetic engineering, but the genetic engineering object in the present invention includes, but is not limited to, the above gene locus, and any effect on capsular expression.
  • the gene can be used as a knock-out modification target in the present invention, and the scope of protection of the present invention should not be limited to the above gene locus.
  • a second object of the present invention is to provide a non-capsulated S. pneumoniae which is a non-capsulated polysaccharide pneumococci which is knocked or modified to affect the capsular polysaccharide synthesis pathway and/or to express related genes. .
  • the gene is selected from the group consisting of one or more of the following genes: cps gene, galU, CcpA and/or PGM.
  • the upstream homologous fragment P1 of the S. pneumoniae galU gene is as shown in SEQ ID NO: 1 of the Sequence Listing, and P2 is represented by SEQ ID NO: 2 of the Sequence Listing.
  • the downstream homologous fragment P3 of the S. pneumoniae galU gene is shown in SEQ ID NO: 3 of the Sequence Listing, and P4 is shown in SEQ ID NO: 4 of the Sequence Listing.
  • the erythromycin resistance gene primer sequence is shown in SEQ ID NO: 5 and SEQ ID NO: 6 of the Sequence Listing.
  • the S. pneumoniae is 18C S. pneumoniae 18C ⁇ galU which knocks out the galU gene.
  • the S. pneumoniae is type 18C Streptococcus pneumoniae ⁇ cps18CE which knocks out the cpsE gene.
  • the S. pneumoniae is an 18C-type S. pneumoniae that simultaneously knocks out the galU gene and the cpsE gene.
  • the S. pneumoniae is Streptococcus pneumoniae 3 ⁇ galU which knocks out the galU gene.
  • the S. pneumoniae is Streptococcus pneumoniae ⁇ cps3D which knocks out the cpsD gene.
  • the S. pneumoniae is Streptococcus pneumoniae type 3 which simultaneously knocks out the galU gene and the cpsD gene.
  • the S. pneumoniae is a 9V type S. pneumoniae 9V ⁇ galU that knocks out the galU gene.
  • the S. pneumoniae is a 9V type S. pneumoniae ⁇ cps9VE that knocks out the cpsE gene.
  • the S. pneumoniae is a 9V type S. pneumoniae which simultaneously knocks out the galU gene and the cpsE gene.
  • the S. pneumoniae is type 19A S. pneumoniae 19A?galU which knocks out the galU gene.
  • the S. pneumoniae is type 19A Streptococcus pneumoniae ⁇ cps19AA which knocks out the cpsA gene.
  • the S. pneumoniae is type 19A Streptococcus pneumoniae ⁇ cps19AG which knocks out the cpsG gene.
  • the S. pneumoniae is a type 19A pneumococci which simultaneously knocks out the galU gene and the cps gene.
  • the cps gene is a cpsA gene and/or a cpsG gene.
  • the S. pneumoniae is Streptococcus pneumoniae 2 ⁇ galU which knocks out the galU gene.
  • the S. pneumoniae is Streptococcus pneumoniae ⁇ cps2K which knocks out the cpsK gene.
  • the S. pneumoniae is Streptococcus pneumoniae type 2 which simultaneously knocks out the galU gene and the cpsK gene.
  • the gene knockout S. pneumoniae of the present invention includes, but is not limited to, the above-mentioned S. pneumoniae, and any S. pneumoniae serotype obtained by knocking out or modifying the genes cps, galU, CcpA and/or PGM It should fall within the scope of protection of the present invention.
  • a third object of the present invention is to provide a method for preparing a non-capsulated S. pneumoniae which knocks out the capsular polysaccharide synthesis genes cps, galU, CcpA and/or S. pneumoniae by gene knockout. PGM.
  • the S. pneumoniae is selected from the group consisting of one or more of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N, 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41, 41F, 42, 43, 44, 45, 46, 47A, 47
  • the gene knockout method is specifically:
  • Design primers design primers based on the upstream and downstream sites affecting the synthesis of the capsular polysaccharide gene of Streptococcus pneumoniae;
  • step b) PCR: using the primers in step a) to amplify the gene fragment, and recovering the corresponding fragment by gel;
  • transformation competent state after culturing the wild bacteria, adding the competent stimulating factor and the fragment recovered by the gel, and culturing to a single colony;
  • step d) Selecting the defective bacteria: picking up the single colonies grown in step c) for expansion culture, and obtaining the defective bacteria having no effect on the synthesis gene of the capsular polysaccharide of Streptococcus pneumoniae.
  • the competent stimulating factor is CSP.
  • the invention also discloses a preparation method of a non-capsular saccharide pneumococci, which uses a knockout method to knock out a gene cps which affects the synthesis of capsular polysaccharide of S. pneumoniae.
  • the invention also discloses a preparation method of the non-capsulated S. pneumoniae, which adopts a gene knockout method to knock out the gene CcpA which affects the synthesis of the capsular polysaccharide of S. pneumoniae.
  • the invention also discloses a preparation method of a non-capsular saccharide pneumococci, which uses a knockout method to knock out a gene PGM which affects the synthesis of capsular polysaccharide of S. pneumoniae.
  • a fourth object of the present invention is to provide a method for knocking out a galU gene of Streptococcus pneumoniae, which is transformed into a competent wild strain after amplification of a galU gene fragment, and cultured to obtain a pneumonia without a galU gene. Streptococcus.
  • the S. pneumoniae is selected from the group consisting of one or more of the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9N , 9V, 10A, 10B, 10C, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B, 12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B , 18C, 18F, 19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A, 25F, 27, 28A, 28F, 29, 31, 32A, 32F , 33A, 33B, 33C, 33D, 33F, 34, 35A, 35B, 35C, 36, 37, 38, 39, 40, 41, 41F, 42, 43, 44, 45, 46, 47A, 47
  • the knockout method of the galU gene is specifically:
  • Design primers design primers based on the upstream and downstream sites of the galU gene
  • step b) PCR: using the primers in step a) to amplify the galU gene fragment, and recovering the corresponding fragment by gel;
  • transformation competent state after culturing the wild bacteria, adding the competent stimulating factor and the fragment recovered by the gel, and culturing to a single colony;
  • step d) Select the defective bacteria: Pick up the single colonies grown in step c) for expansion culture to obtain ⁇ galU-deficient bacteria.
  • the present invention discloses a method for preparing a Streptococcus pneumoniae vaccine, which is obtained by knocking out at least one capsular gene by at least one serotype of S. pneumoniae described in any of the above, and then transforming and screening.
  • Non-capsulated pneumococcal vaccine is obtained by knocking out at least one capsular gene by at least one serotype of S. pneumoniae described in any of the above, and then transforming and screening.
  • the invention also discloses the use of a non-capsulated Streptococcus pneumoniae strain for preparing a Streptococcus pneumoniae vaccine, which is a Streptococcus pneumoniae vaccine antigen.
  • the present invention also provides a Streptococcus pneumoniae antigen which is a non-capsular polysaccharide S. pneumoniae which is knocked or modified to affect the capsular polysaccharide synthesis pathway and/or affects the capsular polysaccharide expression-related gene.
  • the Streptococcus pneumoniae antigen provided in the present invention can be used not only for the development of related immunological preparations of Streptococcus pneumoniae vaccine or Streptococcus pneumoniae, but also for other experiments and tests related to Streptococcus pneumoniae, and combinations and combinations with other antigens. Or conjugation studies.
  • the core of the present invention is how to achieve the immunogenicity study of S. pneumoniae.
  • Each serotype is implemented differently. According to the method provided by the present invention, all pneumonia can be achieved. Effective immunization or treatment of streptococcus.
  • the genetic modification method of the present invention can be used for knocking out or modifying a gene which affects the capsular polysaccharide synthesis pathway and/or affects the capsular polysaccharide expression. That is, the genetic modification method in the present invention is not limited to Streptococcus pneumoniae, and any cocci having a capsular polysaccharide-related gene can be used for the preparation or production of a capsule-free bacterium using the method of the present invention.
  • the present invention successfully achieves the preparation of a non-capsulated Streptococcus pneumoniae by gene knockout, and prepares a vaccine for the preparation of a non-capsulated Streptococcus pneumoniae antigen as an antigen, which can not only realize a whole serotype pneumonia chain.
  • the cocci are immunized and the prepared vaccine is safe and effective.
  • the invention realizes the single or combined knockout of each capsular polysaccharide expression gene locus of each serotype, and obtains a safe, stable and high-efficiency non-capsulated Streptococcus pneumoniae.
  • the preparation method adopts conventional bioengineering means, the process is simple and the product is easy to obtain. Suitable for large-scale preparation and promotion of production.
  • Figure 1 is a graph showing the IgG and IgA antibody titer alignment (18C?galU) of Example 1 of the present invention
  • FIG. 2 is a diagram showing the survival rate (18C type challenge) of mice after immunization with 18C ⁇ galU according to Example 1 of the present invention
  • FIG. 3 is a diagram showing the survival rate of mice (19A type challenge) after immunization with 18C ⁇ galU according to Example 1 of the present invention
  • Figure 4 is a graph showing the titer of IgG and IgA antibody titers (19A?galU) according to Example 2 of the present invention.
  • Figure 5 is a diagram showing the survival rate of 19A?galU immunized mice (type 19A challenge) according to Example 2 of the present invention.
  • Figure 6 is a diagram showing the survival rate (type 1 attack) of 19A?galU immunized mice according to Example 2 of the present invention.
  • Figure 7 is a graph showing an alignment of IgG and IgA antibody titers (9V?galU) according to Example 3 of the present invention.
  • Figure 8 is a diagram showing the survival rate (9V type challenge) of a 9V ⁇ galU immunized mouse according to Example 3 of the present invention.
  • Figure 9 is a diagram showing the survival rate (23F type of challenge) of a 9V ⁇ galU immunized mouse according to Example 3 of the present invention.
  • Figure 10 is a graph showing an alignment of IgG and IgA antibody titers (3?galU) according to Example 4 of the present invention.
  • Figure 11 is a diagram showing the survival rate (type 3 challenge) of 3 ⁇ galU immunized mice according to Example 4 of the present invention.
  • Figure 12 is a graph showing the survival rate (type 14 attack) of 3 ⁇ galU-immunized mice according to Example 4 of the present invention.
  • a pneumococcal vaccine and a preparation method thereof provided by the present invention are further described in detail below in conjunction with the examples.
  • the embodiments described below are illustrative only and are not to be construed as limiting the invention.
  • the galU gene knockout was performed with the highly pathogenic strains 3 (CCUG 6798), 9V (CCUG 36618), 18C (CCUG 7206) and 19A (CCUG 35180).
  • the galU (UTP-glucose-1-phosphate uridylyltransferase, uridine triphosphate-glucose-1-phosphate uridine acyltransferase) gene of Streptococcus pneumoniae is involved in the formation of capsular polysaccharides, usually located outside the cps gene cluster (type 3 Inside, its galU gene is cps3U), which is not a gene necessary for bacterial survival. Choosing this gene as a knockout target can obtain a non-capsulated polysaccharide pneumococci.
  • the knockout target gene and its upstream and downstream sequences can be obtained, and the target fragment can be further designed by PCR amplification, and the gene-deficient strain can be obtained by homologous recombination, so that the capsule can not be obtained.
  • Bacterial genomic DNA extraction kit Bacterial genomic DNA extraction kit, PCR agarose gel recovery kit, plasmid DNA miniprep kit, purchased from TIANGEN, peptone, yeast extract purchased from OXIOD (UK), PCR rTaq enzyme, dNTPs, Buffer, MgCl 2 was purchased from Dalian Bao Biotechnology Co., Ltd., and the competent stimulating peptide (CSP 1 ) was purchased from Nanjing Jinsui.
  • the primers were designed using primer premier 5.0 on the NCBI sequence (No. WP_000202229.1), and the primers were synthesized by Wuhan Jinkaerui.
  • P2 (SEQ ID NO: 2): 5' ATCAAACAAATTTTGGGCCCGG-TCCGTGATAAATAACTTGGTAA 3'
  • P3 (SEQ ID NO: 3): 5'TCGTTAAGGGATCAACTTTGGGA-TTTTCTTTCAACTTCGTCACAT3'
  • P4 (SEQ ID NO: 4): 5'TGCTTTCACTTTATTATCTTGG3'
  • Erythromycin resistance gene primer (Erm)
  • P5 (SEQ ID NO: 5): 5' ATGYGACGAAGAAGTTGAAAGAAAA3'
  • P6 (SEQ ID NO: 6): 5'TTACCAAGTTATTTATCACGGA3'
  • Primers P2 and P3 carry 22 to 23 bases complementary to the 5' and 3' ends of the Erm gene, respectively, such that the amplified upstream and downstream homologous fragments respectively carry a sequence complementary to the Erm gene.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the ratio of the primer to the template was 1:50, and the three PCR-recovered fragments were mixed in an equimolar ratio and used as a template.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the gel recovery kit recovers the PCR product of the expected size and sends it to Wuhan Jinkarui for sequencing verification.
  • S. pneumoniae strain 18C was cultured in C+Y medium to A550 of about 0.1, and 10 ⁇ L of competent stimulating factor CSP (100 ng/ml) was added, and 10 ⁇ L of the ligated PCR product (100 ng/ml) was added to the gel bath at 37 ° C. After 90 min, plate was plated on TSA plates containing erythromycin 0.25 mg/L, and cultured at 37 ° C for one to two days to grow single colonies.
  • a single colony culture was picked and the genome was extracted, and the insert was identified by PCR, and the gel was recovered and verified by sequencing. If the verification is correct, it is a defective bacteria.
  • the correct single colonies were cultured in C+Y medium. When the bacterial density reached about A550 of about 0.2, glycerin was added, mixed and stored in a -80 °C refrigerator.
  • mice Female Balb/c mice were randomly divided into two groups, a group of 12 and a group of 13 rats.
  • S. pneumoniae wild type 18C and galU-deficient strains were diluted to 5 ⁇ 10 8 CFU/ml with PBS, 100 ⁇ L was injected into the peritoneal cavity of mice, the survival state of the mice was observed, and the death time of the mice was recorded, and Mann-Whitney was used. U test for analysis of results. Experimental results: The half lethal time of wild bacteria was hour, while the half death time of the defective strain was day, indicating that the virulence of the galU-deficient strain was significantly decreased.
  • 18C ⁇ galU-deficient bacteria were cultured in C+Y, and wild bacteria from 18C to A620 were about 0.4, 5000 g. The bacteria were collected by centrifugation at 10 min, and immediately after the supernatant was discarded, 1 ml of glutaraldehyde fixative was added along the wall. Under electron microscope, it can be seen that the capsule of the defective bacteria is thinner and sparse than the 18C wild bacteria.
  • the 23-valent polysaccharide vaccine was purchased from the Chengdu Institute of Biological Products, the 13-valent combination vaccine was purchased from the company, the CT adjuvant was purchased from SIGMA, the aluminum adjuvant was purchased from Thermo, and the HRP-labeled goat anti-human IgG was purchased from Wuhan Dr. Sheep anti-human IgA was purchased from Wuhan Dr. De.
  • Balb/c mice were randomly divided into four groups.
  • the first group of positive control PCs (positive control) were 23 groups of 23-valent polysaccharide vaccine and 13-valent combination vaccine, 10 in each group; the second largest group was negative control NC ( Negative control) for CT adjuvant plus PBS in a total of 5 groups, 12 in each group; the third largest group of subcutaneous group was 18C ⁇ galU + aluminum adjuvant in 5 groups, 12 in each group; the fourth group of intranasal immunization IN
  • the (intranasal) group consisted of 18 C ⁇ galU+CT adjuvants in 5 groups of 12 animals each.
  • PC group 2 was intraperitoneally injected with 0.1 ml of 23-valent polysaccharide vaccine, 1 group was intraperitoneally injected with 0.1 ml of 13-valent conjugate vaccine; NC group 5 group was intranasally 30 ⁇ l CT (1 ⁇ g) + PBS; SC group 5 groups were 100 ⁇ l of 10 8 cfu 18C ⁇ galU+100 ⁇ l of aluminum adjuvant was injected subcutaneously; 30 groups of IN group 5 were intranasally administered with 30 ⁇ l of cfu 18C ⁇ galU+CT (1 ⁇ g).
  • Blocking 2% BSA in blocking solution was dissolved in PBST (0.1% Tween ⁇ 20), blocked at 300 ⁇ l for 2 h, and washed three times.
  • Antibody Serum is diluted 1:100, 1:200, 1:400, ...; saliva is diluted 1:25, 1:50, 1:100, .... 100 ⁇ l per well, 37 ° C for 45 min, wash 6 times.
  • Secondary antibody The secondary antibody was diluted 1:5000, 100 ⁇ l per well, and incubated at 37 ° C for 15 min. 50 ⁇ l of stop solution was added to each well to develop an absorbance value (A450) at a wavelength of 450 nm.
  • the antibody titer is defined as the maximum serum dilution factor when the light absorption ratio of the experimental group and the negative control group is greater than or equal to 2.1.
  • the challenge strains were wild type 18C and domestically popular type 19A. There were two models using 18C.
  • the sepsis model was challenged with 3LD50 18C intraperitoneal cavity, the pneumonia model was challenged with 1 ⁇ 10 8 CFU 18C nasal drops, and the 19A type was challenged with 1 ⁇ 10 8 CFU nasal drops.
  • the survival state of the mice was observed for 21 consecutive days, and the survival rate of the mice was calculated.
  • the survival rate of the SC group reached 100% before the 13th, and decreased to 90% from the 13th, and the average survival rate on the 21st was more than 75%. It is almost similar to the protective effect of the 23-valent polysaccharide vaccine (PC group) and the 13-valent conjugate vaccine. There was no statistical difference, and the survival rate of the NC group decreased to 0 within 7 days, indicating that the activity of the experimental bacteria was normal.
  • Figure 3 shows the protective effect of the vaccine against the domestic strain 19A.
  • the survival rate of the SC group and the IN group is significantly higher than that of the negative control NC group, wherein the survival rate of the IN group can reach 100%, and the survival rate of the SC group within 7 days.
  • the 10-day survival rate fell to 90%, the 21-day average survival rate can reach more than 80%, the PC group's 5-day survival rate dropped to 90%, every 3 - 5 days to 10%, 21 days
  • the survival rate was less than 75%, indicating that the survival rate of the IN group was significantly higher than that of the PC group, that is, the protective effect was significantly better than the 23-valent polysaccharide pneumococcal vaccine, and the survival rate of the NC group fell to 0 within 7 days, indicating that the experimental bacteria had normal activity.
  • the above results demonstrate that the attenuated S.
  • pneumoniae vaccine without capsular polysaccharide can provide sufficient protection in mice, and also protect mice from other serotypes of Streptococcus pneumoniae, protection by intranasal immunization. The effect is better than the currently marketed 23-valent pneumococcal polysaccharide vaccine.
  • primers were designed using primer premier 5.0, and the primers were synthesized by Wuhan Jin Kairui.
  • P2 (SEQ ID NO: 2): 5' ATCAAACAAATTTTGGGCCCGG-TCCGTGATAAATAACTTGGTAA 3'
  • P3 (SEQ ID NO: 3): 5'TCGTTAAGGGATCAACTTTGGGA-TTTTCTTTCAACTTCGTCACAT3'
  • P4 (SEQ ID NO: 4): 5'TGCTTTCACTTTATTATCTTGG3'
  • Erythromycin resistance gene primer (Erm)
  • P5 (SEQ ID NO: 5): 5' ATGYGACGAAGAAGTTGAAAGAAAA3'
  • P6 (SEQ ID NO: 6): 5'TTACCAAGTTATTTATCACGGA3'
  • Primers P2 and P3 carry 22 to 23 bases complementary to the 5' and 3' ends of the Erm gene, respectively, such that the amplified upstream and downstream homologous fragments respectively carry a sequence complementary to the Erm gene.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the ratio of the primer to the template was 1:50, and the three PCR-recovered fragments were mixed in an equimolar ratio and used as a template.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the gel recovery kit recovers the PCR product of the expected size and sends it to Wuhan Jinkarui for sequencing verification.
  • S. pneumoniae strain 19A was cultured in C+Y medium to A550 of about 0.1, and 10 ⁇ L of competent stimulating factor CSP (100 ng/ml) was added, and 10 ⁇ L of the ligated PCR product (100 ng/ml) was added to the gel bath at 37 ° C. After 90 min, plate was plated on TSA plates containing erythromycin 0.25 mg/L, and cultured at 37 ° C for one to two days to grow single colonies.
  • a single colony culture was picked and the genome was extracted, and the insert was identified by PCR, and the gel was recovered and verified by sequencing. If the verification is correct, it is a defective bacteria.
  • the correct single colonies were cultured in C+Y medium. When the bacterial density reached about A550 of about 0.2, glycerin was added, mixed and stored in a -80 °C refrigerator.
  • mice Female Balb/c mice were randomly divided into two groups, a group of 12 and a group of 13 rats.
  • S. pneumoniae wild type 19A and galU-deficient strains were diluted to 5 ⁇ 10 8 CFU/ml with PBS, 100 ⁇ L was injected into the peritoneal cavity of mice, the survival state of the mice was observed, and the death time of the mice was recorded, and Mann-Whitney was used. U test for analysis of results. Experimental results: The half lethal time of wild bacteria was hour, while the half death time of the defective strain was day, indicating that the virulence of the galU-deficient strain was significantly decreased.
  • 19A ⁇ galU-deficient bacteria were cultured in C+Y, and wild bacteria from 19A to A620 were about 0.4, 5000 g. The bacteria were collected by centrifugation at 10 min, and 1 ml of glutaraldehyde fixative was added along the wall immediately after the supernatant was discarded. Under electron microscope, it can be seen that the capsule of the defective bacteria is thinner and sparse than that of the 19A wild bacteria.
  • the 23-valent polysaccharide vaccine was purchased from the Chengdu Institute of Biological Products, the 13-valent combination vaccine was purchased from the company, the CT adjuvant was purchased from SIGMA, the aluminum adjuvant was purchased from Thermo, and the HRP-labeled goat anti-human IgG was purchased from Wuhan Dr. Sheep anti-human IgA was purchased from Wuhan Dr. De.
  • Balb/c mice were randomly divided into four groups.
  • the first group of positive control PCs positive control
  • the second largest group was negative control NC ( Negative control) was performed in 5 groups of CT adjuvant plus PBS in groups of 12
  • the third group of subcutaneous group was 19A ⁇ galU+ aluminum adjuvant in 5 groups of 12 groups
  • the fourth group was intranasal immunization IN.
  • the (intranasal) group consisted of 19 A ⁇ galU+CT adjuvants in 5 groups of 12 animals each.
  • PC group 2 was intraperitoneally injected with 0.1 ml of 23-valent polysaccharide vaccine, 1 group was intraperitoneally injected with 0.1 ml of 13-valent conjugate vaccine; NC group 5 group was intranasally 30 ⁇ l CT (1 ⁇ g) + PBS; SC group 5 groups were 100 ⁇ l of 10 8 cfu 19A ⁇ galU + 100 ⁇ l of aluminum adjuvant was injected subcutaneously; 30 groups of IN group 5 were intranasally administered with 30 ⁇ l of cfu 19A ⁇ galU+CT (1 ⁇ g).
  • mice of the tail vein blood and saliva were collected one week after the last immunization, in which saliva was promoted by intraperitoneal injection of 30 ⁇ l of carmicolin. Serum was separated, and the level of salivary protein was adjusted to the same level with PBS. After appropriate dilution, the antibody titer was determined by ELISA. The specific steps are as follows:
  • Blocking 2% BSA in blocking solution was dissolved in PBST (0.1% Tween ⁇ 20), blocked at 300 ⁇ l for 2 h, and washed three times.
  • Antibody Serum is diluted 1:100, 1:200, 1:400, ...; saliva is diluted 1:25, 1:50, 1:100, .... 100 ⁇ l per well, 37 ° C for 45 min, wash 6 times.
  • Secondary antibody The secondary antibody was diluted 1:5000, 100 ⁇ l per well, and incubated at 37 ° C for 15 min. 50 ⁇ l of stop solution was added to each well to develop an absorbance value (A450) at a wavelength of 450 nm.
  • the antibody titer is defined as the maximum serum dilution factor when the light absorption ratio of the experimental group and the negative control group is greater than or equal to 2.1.
  • the challenge strains were wild type 19A and domestically popular type 1. There were two models with 19A.
  • the sepsis model was challenged with 3LD50 19A intraperitoneal cavity.
  • the pneumonia model was challenged with 1 ⁇ 10 8 CFU 19A nasal drops; type 1 was challenged with 1 ⁇ 10 8 CFU nasal drops.
  • the survival state of the mice was observed for 21 consecutive days, and the survival rate of the mice was calculated.
  • the average survival rate on the 21st was more than 75%.
  • the protection effect was similar to that of the 23-valent polysaccharide vaccine (PC group) and the 13-valent combination vaccine. Learning differences, the survival rate of the NC group fell to 0 within 7 days, indicating that the experimental bacteria activity was normal.
  • Figure 6 illustrates the protective effect of the vaccine on the domestic strain 1 of the domestic strain.
  • the survival rate of the SC group and the IN group was significantly higher than that of the negative control NC group, and the 21-day survival rate of the IN group was 100%, and the SC group 7
  • the daily survival rate can reach 100%, and it will drop to 90% from the 9th.
  • the average survival rate on the 21st can reach 80%.
  • the survival rate of the PC group will drop to 90% on the 5th, and will drop by 10% every 3 ⁇ 5 days.
  • the survival rate on the 21st was less than 75%, indicating that the survival rate of the IN group was significantly higher than that of the PC group, that is, the protective effect was significantly better than the 23-valent polysaccharide pneumococcal vaccine, and the survival rate of the NC group fell to 0 within 7 days, indicating the experimental bacteria.
  • the activity is normal.
  • the above results demonstrate that the attenuated S. pneumoniae vaccine without capsular polysaccharide can provide sufficient protection in mice, and also protect mice from other serotypes of Streptococcus pneumoniae, protection by intranasal immunization. The effect is better than the currently marketed 23-valent pneumococcal polysaccharide vaccine.
  • primers were designed using primer premier 5.0 on the NCBI sequence (No. NZ_MAVR01000057.1), and the primers were synthesized by Wuhan Jinkaerui.
  • P2 (SEQ ID NO: 2): 5' ATCAAACAAATTTTGGGCCCGG-TCCGTGATAAATAACTTGGTAA 3'
  • P3 (SEQ ID NO: 3): 5'TCGTTAAGGGATCAACTTTGGGA-TTTTCTTTCAACTTCGTCACAT3'
  • P4 (SEQ ID NO: 4): 5'TGCTTTCACTTTATTATCTTGG3'
  • Erythromycin resistance gene primer (Erm)
  • P5 (SEQ ID NO: 5): 5' ATGYGACGAAGAAGTTGAAAGAAAA3'
  • P6 (SEQ ID NO: 6): 5'TTACCAAGTTATTTATCACGGA3'
  • Primers P2 and P3 carry 22 to 23 bases complementary to the 5' and 3' ends of the Erm gene, respectively, such that the amplified upstream and downstream homologous fragments respectively carry a sequence complementary to the Erm gene.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the ratio of the primer to the template was 1:50, and the three PCR-recovered fragments were mixed in an equimolar ratio and used as a template.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the gel recovery kit recovers the PCR product of the expected size and sends it to Wuhan Jinkarui for sequencing verification.
  • S. pneumoniae strain 9V was cultured in C+Y medium to A550 of about 0.1, and 10 ⁇ L of competent stimulating factor CSP (100 ng/ml) was added, and 10 ⁇ L of the linked PCR product (100 ng/ml) was added to the gel, and the water bath was at 37 ° C. After 90 min, plate was plated on TSA plates containing erythromycin 0.25 mg/L, and cultured at 37 ° C for one to two days to grow single colonies.
  • a single colony culture was picked and the genome was extracted, and the insert was identified by PCR, and the gel was recovered and verified by sequencing. If the verification is correct, it is a defective bacteria.
  • the correct single colonies were cultured in C+Y medium. When the bacterial density reached about A550 of about 0.2, glycerin was added, mixed and stored in a -80 °C refrigerator.
  • mice Female Balb/c mice were randomly divided into two groups, a group of 12 and a group of 13 rats.
  • S. pneumoniae wild type 9V and galU-deficient strains were diluted to 5 ⁇ 10 8 CFU/ml with PBS, 100 ⁇ L was injected into the peritoneal cavity of mice, the survival state of the mice was observed, and the death time of the mice was recorded, and Mann-Whitney was used. U test for analysis of results. Experimental results: The half lethal time of wild bacteria was hour, while the half death time of the defective strain was day, indicating that the virulence of the galU-deficient strain was significantly decreased.
  • the 23-valent polysaccharide vaccine was purchased from the Chengdu Institute of Biological Products, the 13-valent combination vaccine was purchased from the company, the CT adjuvant was purchased from SIGMA, the aluminum adjuvant was purchased from Thermo, and the HRP-labeled goat anti-human IgG was purchased from Wuhan Dr. Sheep anti-human IgA was purchased from Wuhan Dr. De.
  • Balb/c mice were randomly divided into four groups.
  • the first group of positive control PCs (positive control) were 23 groups of 23-valent polysaccharide vaccine and 13-valent combination vaccine, 10 in each group; the second largest group was negative control NC ( Negative control) was performed in 5 groups of CT adjuvant plus PBS in groups of 12; the third group of subcutaneous group was 9V ⁇ galU+ aluminum adjuvant in 5 groups, 12 in each group; the fourth group was intranasal immunization IN
  • the (intranasal) group consisted of 9 V ⁇ galU+CT adjuvants in 5 groups of 12 animals each.
  • PC group 2 was intraperitoneally injected with 0.1 ml of 23-valent polysaccharide vaccine, 1 group was intraperitoneally injected with 0.1 ml of 13-valent conjugate vaccine; NC group 5 group was intranasally 30 ⁇ l CT (1 ⁇ g) + PBS; SC group 5 groups were 100 ⁇ l of 10 8 cfu 9V ⁇ galU + 100 ⁇ l of aluminum adjuvant was injected subcutaneously; 30 groups of IN group 5 were intranasally administered with 30 ⁇ l of cfu 9V ⁇ galU+CT (1 ⁇ g).
  • a second immunization was performed two weeks later (except for the PC group, which was only immunized once), and the immunization methods of the NC group, the SC group, and the IN group were the same as the first immunization.
  • the third immunization was performed without adjuvant.
  • the immunization dose was as follows: 30 ⁇ l PBS in the NC group; 200 ⁇ l 10 8 cfu 9 V ⁇ galU in the SC group; 30 ⁇ l 10 8 cfu 9 V ⁇ gal in the IN group.
  • mice of the tail vein blood and saliva were collected one week after the last immunization, in which saliva was promoted by intraperitoneal injection of 30 ⁇ l of carmicolin. Serum was separated, and the level of salivary protein was adjusted to the same level with PBS. After appropriate dilution, the antibody titer was determined by ELISA. The specific steps are as follows:
  • Blocking 2% BSA in blocking solution was dissolved in PBST (0.1% Tween ⁇ 20), blocked at 300 ⁇ l for 2 h, and washed three times.
  • Antibody Serum is diluted 1:100, 1:200, 1:400, ...; saliva is diluted 1:25, 1:50, 1:100, .... 100 ⁇ l per well, 37 ° C for 45 min, wash 6 times.
  • Secondary antibody The secondary antibody was diluted 1:5000, 100 ⁇ l per well, and incubated at 37 ° C for 15 min. 50 ⁇ l of stop solution was added to each well to develop an absorbance value (A450) at a wavelength of 450 nm.
  • the antibody titer is defined as the maximum serum dilution factor when the light absorption ratio of the experimental group and the negative control group is greater than or equal to 2.1.
  • the challenge strains were wild type 9V and domestically popular 23F type. There were two models with 9V, the sepsis model was challenged with 3LD50 9V intraperitoneal cavity, the pneumonia model was challenged with 1 ⁇ 10 8 CFU 9V nasal drops, and the 23F type was challenged with 1 ⁇ 10 8 CFU nasal drops. The survival state of the mice was observed for 21 consecutive days, and the survival rate of the mice was calculated.
  • the average survival rate on the 21st was more than 75%.
  • the protection effect was similar to that of the 23-valent polysaccharide vaccine (PC group) and the 13-valent combination vaccine. Learning differences, the survival rate of the NC group fell to 0 within 7 days, indicating that the experimental bacteria activity was normal.
  • Figure 9 shows the protective effect of the vaccine on the domestically-prevalent strain 23F.
  • the survival rate of the SC group and the IN group was significantly higher than that of the negative control NC group, and the 21-day survival rate of the IN group was 100%, SC group 7
  • the daily survival rate can reach 100%, and it will drop to 90% from the 10th.
  • the average survival rate on the 21st can reach 80%.
  • the survival rate of the PC group will drop to 90% on the 5th, and will drop by 10% every 3 ⁇ 5 days.
  • the survival rate on the 21st was less than 75%, indicating that the survival rate of the IN group was significantly higher than that of the PC group, that is, the protective effect was significantly better than the 23-valent polysaccharide pneumococcal vaccine, and the survival rate of the NC group fell to 0 within 7 days, indicating the experimental bacteria.
  • the activity is normal.
  • the above results demonstrate that the attenuated S. pneumoniae vaccine without capsular polysaccharide can provide sufficient protection in mice, and also protect mice from other serotypes of Streptococcus pneumoniae, protection by intranasal immunization. The effect is better than the currently marketed 23-valent pneumococcal polysaccharide vaccine.
  • the primers were designed using primer premier 5.0 on the NCBI sequence (No. NZ_LSLM01000003.1), and the primers were synthesized by Wuhan Jinkarui.
  • P2 (SEQ ID NO: 2): 5' ATCAAACAAATTTTGGGCCCGG-TCCGTGATAAATAACTTGGTAA 3'
  • P3 (SEQ ID NO: 3): 5'TCGTTAAGGGATCAACTTTGGGA-TTTTCTTTCAACTTCGTCACAT3'
  • P4 (SEQ ID NO: 4): 5'TGCTTTCACTTTATTATCTTGG3'
  • Erythromycin resistance gene primer (Erm)
  • P5 (SEQ ID NO: 5): 5' ATGYGACGAAGAAGTTGAAAGAAAA3'
  • P6 (SEQ ID NO: 6): 5'TTACCAAGTTATTTATCACGGA3'
  • Primers P2 and P3 carry 22 to 23 bases complementary to the 5' and 3' ends of the erm gene, respectively, such that the amplified upstream and downstream homologous fragments respectively carry a sequence complementary to the erm gene.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the ratio of the primer to the template was 1:50, and the three PCR-recovered fragments were mixed in an equimolar ratio and used as a template.
  • PCR reaction conditions pre-denaturation at 95 ° C for 5 min, 1 cycle; denaturation at 95 ° C for 1 min, annealing at 55 ° C for 30 s, extension at 72 ° C for 1 min, 30 cycles; extension at 72 ° C for 10 min, 1 cycle.
  • the gel recovery kit recovers the PCR product of the expected size and sends it to Wuhan Jinkarui for sequencing verification.
  • S. pneumoniae type 3 was cultured in C+Y medium to A550 of about 0.1, and 10 ⁇ L of competent stimulating factor CSP (100 ng/ml) was added, and 10 ⁇ L of the ligated PCR product (100 ng/ml) was added to the gel bath at 37 ° C. After 90 min, plate was plated on TSA plates containing erythromycin 0.25 mg/L, and cultured at 37 ° C for one to two days to grow single colonies.
  • a single colony culture was picked and the genome was extracted, and the insert was identified by PCR, and the gel was recovered and verified by sequencing. If the verification is correct, it is a defective bacteria.
  • the correct single colonies were cultured in C+Y medium. When the bacterial density reached about A550 of about 0.2, glycerin was added, mixed and stored in a -80 °C refrigerator.
  • mice Female Balb/c mice were randomly divided into two groups, a group of 12 and a group of 13 rats.
  • S. pneumoniae wild type 3 and galU-deficient strains were diluted to 5 ⁇ 10 8 CFU/ml with PBS, 100 ⁇ L was injected into the peritoneal cavity of mice, the survival state of the mice was observed, and the death time of the mice was recorded, and Mann-Whitney was used. U test for analysis of results. Experimental results: The half lethal time of wild bacteria was hour, while the half death time of the defective strain was day, indicating that the virulence of the galU-deficient strain was significantly decreased.
  • the 23-valent polysaccharide vaccine was purchased from the Chengdu Institute of Biological Products, the 13-valent combination vaccine was purchased from the company, the CT adjuvant was purchased from SIGMA, the aluminum adjuvant was purchased from Thermo, and the HRP-labeled goat anti-human IgG was purchased from Wuhan Dr. Sheep anti-human IgA was purchased from Wuhan Dr. De.
  • Balb/c mice were randomly divided into four groups.
  • the first group of positive control PCs positive control
  • the second largest group was negative control NC ( Negative control) was performed in 5 groups of CT adjuvant plus PBS, 12 in each group;
  • the third group of subcutaneous group was 3 ⁇ galU+ aluminum adjuvant in 5 groups, 12 in each group;
  • the fourth group was intranasal immunization IN
  • the (intranasal) group consisted of 3 ⁇ galU+CT adjuvants in 5 groups of 12 animals each.
  • PC group 2 was intraperitoneally injected with 0.1 ml of 23-valent polysaccharide vaccine, 1 group was intraperitoneally injected with 0.1 ml of 13-valent conjugate vaccine; NC group 5 group was intranasally 30 ⁇ l CT (1 ⁇ g) + PBS; SC group 5 groups were 100 ⁇ l of 10 8 cfu 3 ⁇ galU + 100 ⁇ l of aluminum adjuvant was injected subcutaneously; 30 groups of IN group 5 were intranasally administered with 30 ⁇ l of cfu 3 ⁇ galU+CT (1 ⁇ g).
  • mice of the tail vein blood and saliva were collected one week after the last immunization, in which saliva was promoted by intraperitoneal injection of 30 ⁇ l of carmicolin. Serum was separated, and the level of salivary protein was adjusted to the same level with PBS. After appropriate dilution, the antibody titer was determined by ELISA. The specific steps are as follows:
  • Blocking 2% BSA in blocking solution was dissolved in PBST (0.1% Tween ⁇ 20), blocked at 300 ⁇ l for 2 h, and washed three times.
  • Antibody Serum is diluted 1:100, 1:200, 1:400, ...; saliva is diluted 1:25, 1:50, 1:100, .... 100 ⁇ l per well, 37 ° C for 45 min, wash 6 times.
  • Secondary antibody The secondary antibody was diluted 1:5000, 100 ⁇ l per well, and incubated at 37 ° C for 15 min. 50 ⁇ l of stop solution was added to each well to develop an absorbance value (A450) at a wavelength of 450 nm.
  • the antibody titer is defined as the maximum serum dilution factor when the light absorption ratio of the experimental group and the negative control group is greater than or equal to 2.1.
  • the challenge strains were wild type 3 and domestically popular type 14. There were two models of type 3, the model of sepsis was challenged with 3LD50 type 3 abdominal cavity, the model of pneumonia was challenged with type 1 nasal drops of 1 ⁇ 10 8 CFU, and the type 14 was challenged with 1 ⁇ 10 8 CFU. The survival state of the mice was observed for 21 consecutive days, and the survival rate of the mice was calculated.
  • the average survival rate on the 21st was more than 75%.
  • the protection effect was similar to that of the 23-valent polysaccharide vaccine (PC group) and the 13-valent conjugate vaccine.
  • the survival rate of the NC group decreased to 0 within 7 days, indicating that the activity of the experimental bacteria was normal.
  • Figure 12 is a diagram showing the protective effect of the vaccine on the domestically-prevalent strain type 14.
  • the survival rate of the SC group and the IN group was significantly higher than that of the negative control NC group, and the 21-day survival rate of the IN group was 100%, and the SC group 7
  • the daily survival rate can reach 100%, and it will drop to 90% from the 8th.
  • the average survival rate on the 21st can reach 80%.
  • the survival rate of the PC group will drop to 90% on the 5th, and will drop by 10% every 3 ⁇ 5 days.
  • the survival rate on the 21st was less than 75%, indicating that the survival rate of the IN group was significantly higher than that of the PC group, that is, the protective effect was significantly better than the 23-valent polysaccharide pneumococcal vaccine, and the survival rate of the NC group fell to 0 within 7 days, indicating the experimental bacteria.
  • the activity is normal.
  • the above results demonstrate that the attenuated S. pneumoniae vaccine without capsular polysaccharide can provide sufficient protection in mice, and also protect mice from other serotypes of Streptococcus pneumoniae, protection by intranasal immunization. The effect is better than the currently marketed 23-valent pneumococcal polysaccharide vaccine.

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Abstract

本发明公开了一种肺炎链球菌疫苗及其制备方法，其中，所述肺炎链球菌疫苗包括至少一个血清型的肺炎链球菌为抗原，所述肺炎链球菌为经敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因后的无荚膜多糖肺炎链球菌。本发明采用基因敲除法成功实现了无荚膜多糖的肺炎链球菌的制备，以制备出的无荚膜多糖肺炎链球菌作为抗原制备疫苗，不仅可以实现全血清型的肺炎链球菌免疫，且制得的疫苗安全有效，本发明实现了各血清型中影响荚膜多糖生成的各基因位点的单独或联合敲除，获得了安全稳定高效的无荚膜多糖肺炎链球菌，制备方法采用常规生物工程手段，过程简单且产品易得，适合大规模制备推广生产。

Description

一种肺炎链球菌疫苗及其制备方法 技术领域
本发明涉及一种肺炎链球菌疫苗及其制备方法，属于生物制药领域。
背景技术
肺炎链球菌(Streptococcus pneumoniae)，寄居于正常人的鼻咽腔中，是细菌性大叶性肺炎、脑膜炎、中耳炎、肺炎、支气管炎的主要病原菌。肺炎链球菌导致的疾病一直是全球严重的公共卫生问题，在全世界范围内有较高的发病率和病死率，尤其是对2岁以下的儿童和老人。目前已上市的肺炎链球菌荚膜糖疫苗和荚膜糖蛋白质结合疫苗，其设计都基于肺炎链球菌荚膜糖，涵盖了导致肺炎链球菌性疾病的最常见血清型。但肺炎链球菌荚膜糖为胸腺非依赖性抗原(Thymus independent antigen，TI‐Ag)，抗体反应主要依赖于其重复单位组成的线性表位，在无T淋巴细胞辅助的情况下直接与B淋巴细胞表面的IgM受体交联，所诱导的抗体主要为IgM和IgG2，缺少较好的补体活化能力，抗体水平不能维持足够长的时间，且不能诱导免疫记忆，无法在2岁以下幼儿中产生免疫保护。荚膜糖复杂的结构导致每一个血清型的免疫原性不同，无法产生有效的免疫应答。肺炎链球菌结合疫苗包括血清型别多，各型别用于结合的特异性结构不同，导致其每个型别的结合方法相异。对荚膜糖的修饰及与载体蛋白的结合要在保证荚膜糖特异基团不丢失、抗原性和免疫原性不受影响的前提下进行，同时为了避免糖链的过度交联和结合物除菌过滤的要求，对荚膜糖及结合物分子的大小应当有一定控制。7价疫苗于2000年2月在美国获准使用。由于肺炎链球菌型别多，在结合疫苗的制作过程需要结合蛋白成分，因蛋白成分可以引起局部反应，所以生产包含12个型别以上的结合疫苗就很困难。结合疫苗在初次免疫后活的抗体浓度仅能维持几个月，随后就会下降到免疫前水平；并且结合疫苗的整个工艺过程中需要加入多种化学试剂参与反应，并且荚膜糖蛋白结合疫苗的血清型覆盖率低和非疫苗血清型肺炎链球菌感染性疾病的增加使得更多研究者开始关注其他方向的肺炎链球菌疫苗开发。
肺炎链球菌由于其血清型多，导致其抗原本身的抗原结构大稳定性差，难以与其他疫苗联合共存使用，并且现有的肺炎链球菌荚膜糖蛋白结合疫苗均用白喉或破伤风类毒素作为蛋白载体，这种疫苗的主要成分为血清型特异性的荚膜糖，对未包含在疫苗内的其他血清型肺炎链球菌感染无效，即缺乏交叉免疫保护效果；并将与已在儿童常规免疫接种中使用的白喉及破伤风疫苗产生干扰，破坏现有的免疫效果。因此研究一款能够跨越血清型肺炎链球菌蛋白疫苗成为了疫苗研发领域的当务之急。
目前技术人员主要尝试的全血清型免疫方向为蛋白疫苗方向，即提取肺炎链球菌中可以激起体内免疫应答的稳定蛋白作为抗原进行免疫反应，但蛋白不仅体外活性大大降低，且会引起较为强烈的免疫副作用，对于耐受力较差的婴幼儿来说免疫过程痛感过强。蛋白疫苗与肺炎链球菌菌体的理化性质差异巨大，虽有科研机构表示已有研发成功的肺炎链球菌蛋白疫苗，但目前还没有完全获批上市的品种，说明这一方向还需要一定的临床论证过程。
肺炎链球菌主要的致病因子是荚膜，荚膜主要的化学成分为荚膜多糖，近年来随着研究的不断深入，发现无荚膜的肺炎链球菌裸菌在活体状态下即可激起体内免疫应答，并且由于无荚膜多糖，因此不会致病，也不用区分不同血清型，是新一代安全有效的肺炎链球菌疫苗的发展新方向。荚膜多糖的表达涉及一系列复杂的生化过程，其中阻断荚膜多糖表达中的任何一步，或对表达过程中的关键酶、关键反应物进行干扰，以影响相关的荚膜多糖表达或合成步骤，都会影响荚膜多糖最终的形成。因此申请人以此为科研攻关重点，设计了一种通过基因工程手段产生无荚膜的肺炎链球菌作为抗原的疫苗及该疫苗的制备方 法。
发明内容
针对现有技术存在的上述问题，本发明的目的是获得一种肺炎链球菌疫苗及其制备方法。
为实现上述发明目的之一，本发明采用的肺炎链球菌疫苗的技术方案如下：
所述肺炎链球菌疫苗包括至少一个血清型的肺炎链球菌为抗原，所述肺炎链球菌为经敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因后的无荚膜多糖肺炎链球菌。敲除或修饰任何一个或以上的荚膜多糖表达或合成途径中的相关基因，均可以对整个荚膜多糖的表达或合成途径产生影响，即影响最终荚膜多糖的形成。换言之，只要是可以影响荚膜多糖形成的基因，均可以作为本发明中敲除或修饰的对象。
所述“基因敲除或修饰”是指通过一定的途径使机体特定的基因失活或缺失的技术。任何可以进行基因失活或缺失的生物学方法均可以作为本发明中的“基因敲除或修饰”方法使用。
优选的，所述肺炎链球菌选自以下血清型中的一种或以上的组合：1、2、3、4、5、6A、6B、6C、7A、7B、7C、7F、8、9A、9L、9N、9V、10A、10B、10C、10F、11A、11B、11C、11D、11F、12A、12B、12F、13、14、15A、15B、15C、15F、16A、16F、17A、17F、18A、18B、18C、18F、19A、19B、19C、19F、20、21、22A、22F、23A、23B、23F、24A、24B、24F、25A、25F、27、28A、28F、29、31、32A、32F、33A、33B、33C、33D、33F、34、35A、35B、35C、36、37、38、39、40、41、41F、42、43、44、45、46、47A、47F和/或48。
肺炎链球菌目前据报道共91个血清型，虽然仅有少数血清型致病，但各血清型均具有基本的荚膜结构，可以作为本发明中的无荚膜肺炎链球菌疫苗的抗原改性来源。
优选的，所述肺炎链球菌疫苗为单血清型肺炎链球菌疫苗。由于无荚膜肺炎链球菌疫苗具有突破血清型限制的特点，因此单血清型改性即可以起到良好的免疫效果。
各致病血清型为改性的优选，所述肺炎链球菌选自以下血清型中的一种：1、2、3、4、5、6A、6B、7F、8、9N、9V、10A、11A、12F、14、15B、17F、18C、19A、19F、20、22F、23F或33F。
所述基因选自以下基因中的一种或以上的组合：cps基因、galU、CcpA和/或PGM。
优选的，cps基因选自以下基因中的一种或以上的组合：cpsA、cpsB、cpsC、cpsD、CpsE、CpsF、CpsG和/或CpsI。
CcpA可能与肺炎链球菌荚膜多糖(CPS)基因座启动子区域结合，且CcpA(catabolite control protein A，CcpA)是一种与糖代谢相关的转录调控因子，调控了肺炎链球菌多种基因的表达；葡萄糖磷酸变位酶(PGM)、galU(尿苷三磷酸‐葡萄糖‐1‐磷酸尿苷酰基转移酶)、上述基因位点可以单独或联合敲除，以制备无荚膜或荚膜缺失的肺炎链球菌。
本申请以目前已证实具有荚膜多糖合成作用的基因作为基因工程的客体进行敲除或修饰，但本发明中的基因工程客体包括但不限于上述基因位点，任何对于荚膜表达具有作用的基因均可作为本发明中的敲除改性对象，本发明的保护范围不应限于上述基因位点。
本发明的第二个目的在于提供一种无荚膜肺炎链球菌，所述肺炎链球菌为经敲除或修饰影响荚膜多糖合成途径和/或表达相关基因后的无荚膜多糖肺炎链球菌。
优选的，所述基因选自以下基因中的一种或以上的组合：cps基因、galU、CcpA和/或PGM。
优选的，所述的肺炎链球菌galU基因的上游同源片段P1如序列表SEQ ID NO：1所示，P2如序列表SEQ ID NO：2所示。所述的肺炎链球菌galU基因的下游同源片段P3如序列表SEQ ID NO：3所示，P4如序列表SEQ ID NO：4所示。红霉素抗性基因引物序列如序列表SEQ ID NO：5和SEQ ID NO：6所示。
作为一种优选的实施方式，所述肺炎链球菌为敲除galU基因的18C肺炎链球菌18CΔgalU。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsE基因的18C型肺炎链球菌Δcps18CE。
本发明的另一种优选方式中，所述肺炎链球菌为同时敲除galU基因和cpsE基因的18C型肺炎链球菌。
本发明的另一种优选方式中，所述肺炎链球菌为敲除galU基因的3型肺炎链球菌3ΔgalU。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsD基因的3型肺炎链球菌Δcps3D。
本发明的另一种优选方式中，所述肺炎链球菌为同时敲除galU基因和cpsD基因的3型肺炎链球菌。
本发明的另一种优选方式中，所述肺炎链球菌为敲除galU基因的9V型肺炎链球菌9VΔgalU。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsE基因的9V型肺炎链球菌Δcps9VE。
本发明的另一种优选方式中，所述肺炎链球菌为同时敲除galU基因和cpsE基因的9V型肺炎链球菌。
本发明的另一种优选方式中，所述肺炎链球菌为敲除galU基因的19A型肺炎链球菌19AΔgalU。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsA基因的19A型肺炎链球菌Δcps19AA。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsG基因的19A型肺炎链球菌Δcps19AG。
本发明的另一种优选方式中，所述肺炎链球菌为同时敲除galU基因和cps基因的19A型肺炎链球菌。其中所述cps基因为cpsA基因和/或cpsG基因。
本发明的另一个优选方式中，所述肺炎链球菌为敲除galU基因的2型肺炎链球菌2ΔgalU。
本发明的另一种优选方式中，所述肺炎链球菌为敲除cpsK基因的2型肺炎链球菌Δcps2K。
本发明的另一种优选方式中，所述肺炎链球菌为同时敲除galU基因和cpsK基因的2型肺炎链球菌。
本发明中的基因敲除的肺炎链球菌包括但不限于上述肺炎链球菌，任何肺炎链球菌血清型经过敲除或修饰基因cps、galU、CcpA和/或PGM后得到的缺陷型肺炎链球菌均应落入本发明的保护范围之内。
本发明的第三个目的在于提供一种无荚膜糖肺炎链球菌的制备方法，所述方法采用基因敲除的方法敲除肺炎链球菌的荚膜多糖合成基因cps、galU、CcpA和/或PGM。
优选的，所述肺炎链球菌选自以下血清型中的一种或以上的组合：1、2、3、4、5、6A、6B、6C、7A、7B、7C、7F、8、9A、9L、9N、9V、10A、10B、10C、10F、11A、11B、11C、11D、11F、12A、12B、12F、13、14、15A、15B、15C、15F、16A、16F、17A、17F、18A、18B、18C、18F、19A、19B、19C、19F、20、21、22A、22F、23A、23B、23F、24A、24B、24F、25A、25F、27、28A、28F、29、31、32A、32F、33A、33B、33C、33D、33F、34、35A、35B、35C、36、37、38、39、40、41、41F、42、43、44、45、46、47A、47F和/或48。
优选的，基因敲除方法具体为：
a)设计引物：根据影响肺炎链球菌荚膜多糖合成基因的上下游位点，设计引物；
b)PCR：采用步骤a)中的引物扩增基因片段，胶回收对应片段；
c)转化感受态：培养野生菌后加入感受态刺激因子及胶回收的片段，培养至单菌落；
d)选取缺陷菌：挑取步骤c)中长出的单菌落进行扩大培养，获得无影响肺炎链球菌荚膜多糖合成基因的缺陷菌。
优选的，所述感受态刺激因子为CSP。
本发明还公开了一种无荚膜糖肺炎链球菌的制备方法，所述方法采用基因敲除的方法敲除影响肺炎链球菌荚膜多糖合成的基因cps。
本发明还公开了一种无荚膜糖肺炎链球菌的制备方法，所述方法采用基因敲除的方法敲除影响肺炎链球菌荚膜多糖合成的基因CcpA。
本发明还公开了一种无荚膜糖肺炎链球菌的制备方法，所述方法采用基因敲除的方法敲除影响肺炎链球菌荚膜多糖合成的基因PGM。
本发明的第四个目的在于提供一种肺炎链球菌galU基因的敲除方法，所述敲除方法在扩增galU基因片段后转化至感受态的野生菌中，筛选培养获得无galU基因的肺炎链球菌。
所述肺炎链球菌选自以下血清型中的一种或以上的组合：1、2、3、4、5、6A、6B、6C、7A、7B、7C、7F、8、9A、9L、9N、9V、10A、10B、10C、10F、11A、11B、11C、11D、11F、12A、12B、12F、13、14、15A、15B、15C、15F、16A、16F、17A、17F、18A、18B、18C、18F、19A、19B、19C、19F、20、21、22A、22F、23A、23B、23F、24A、24B、24F、25A、25F、27、28A、28F、29、31、32A、32F、33A、33B、33C、33D、33F、34、35A、35B、35C、36、37、38、39、40、41、41F、42、43、44、45、46、47A、47F和/或48。
当敲除基因为galU基因时，所述galU基因的敲除方法具体为：
a)设计引物：根据galU基因的上下游位点，设计引物；
b)PCR：采用步骤a)中的引物扩增galU基因片段，胶回收对应片段；
c)转化感受态：培养野生菌后加入感受态刺激因子及胶回收的片段，培养至单菌落；
d)选取缺陷菌：挑取步骤c)中长出的单菌落进行扩大培养，获得ΔgalU缺陷菌。
综上，本发明公开了一种肺炎链球菌疫苗的制备方法，所述方法以上述任一项所述的至少一种血清型的肺炎链球菌经敲除至少一个荚膜基因后，转化筛选得到无荚膜肺炎链球菌疫苗。
本发明还公开了一种无荚膜肺炎链球菌在制备肺炎链球菌疫苗中的应用，所述无荚膜肺炎链球菌为肺炎链球菌疫苗抗原。
本发明还提供了一种肺炎链球菌抗原，所述肺炎链球菌抗原为经敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因后的无荚膜多糖肺炎链球菌。
本发明中提供的肺炎链球菌抗原不仅可以用于肺炎链球菌疫苗或肺炎链球菌的相关免疫制剂的研发，还可以用于其他的肺炎链球菌相关实验、测试，以及与其他抗原的联合、组合或缀合研究。
值得一提的是，本发明提供的核心要义在于如何实现无荚膜肺炎链球菌的免疫原性研究，每个血清型的实现方法均有不同，根据本发明提供的方法，可实现所有的肺炎链球菌的有效免疫或治疗。
本发明中的基因修饰方法，可用于敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因。也就是说，本发明中的基因修饰方法不仅限于肺炎链球菌，任何具有荚膜多糖相关基因的球菌均可使用本发明中的方法进行无荚膜菌的制备或生产。
与现有技术相比，本发明采用基因敲除法成功实现了无荚膜的肺炎链球菌的制备，以制备出的无荚膜肺炎链球菌作为抗原制备疫苗，不仅可以实现全血清型的肺炎链球菌免疫，且制得的疫苗安全有效。本发明实现了各血清型的各荚膜多糖表达基因位点的单独或联合 敲除，获得了安全稳定高效的无荚膜肺炎链球菌，制备方法采用常规生物工程手段，过程简单且产品易得，适合大规模制备推广生产。
附图说明
图1为本发明实施例1的IgG和IgA抗体滴度比对(18CΔgalU)图；
图2为本发明实施例1的18CΔgalU免疫后小鼠生存率(18C型攻毒)图；
图3为本发明实施例1的18CΔgalU免疫后小鼠生存率(19A型攻毒)图；
图4为本发明实施例2的IgG和IgA抗体滴度比对(19AΔgalU)图；
图5为本发明实施例2的19AΔgalU免疫小鼠生存率(19A型攻毒)图；
图6为本发明实施例2的19AΔgalU免疫小鼠生存率(1型攻毒)图；
图7为本发明实施例3的IgG和IgA抗体滴度比对(9VΔgalU)图；
图8为本发明实施例3的9VΔgalU免疫小鼠生存率(9V型攻毒)图；
图9为本发明实施例3的9VΔgalU免疫小鼠生存率(23F型攻毒)图；
图10为本发明实施例4的IgG和IgA抗体滴度比对(3ΔgalU)图；
图11为本发明实施例4的3ΔgalU免疫小鼠生存率(3型攻毒)图；
图12为本发明实施例4的3ΔgalU免疫小鼠生存率(14型攻毒)图。
具体实施方式
下面结合实施例对本发明提供的一种肺炎链球菌疫苗及其制备方法作进一步详细、完整地说明。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验材料如无特殊说明，均为市场购买得到并按说明书操作。
本实施例以致病性较强的流行菌株3(CCUG 6798)、9V(CCUG 36618)、18C(CCUG 7206)和19A(CCUG 35180)进行galU基因敲除。肺炎链球菌的galU(UTP–glucose‐1‐phosphate uridylyltransferase，尿苷三磷酸‐葡萄糖‐1‐磷酸尿苷酰基转移酶)基因参与荚膜多糖的形成，一般位于cps基因簇之外(3型在内，其galU基因是cps3U)，不是细菌生存所必须的基因，选择此基因作为敲除对象，可以获得无荚膜多糖肺炎链球菌。
根据NCBI上记录的序列信息(非全基因组序列)，可获得敲除目标基因及其上游和下游序列，进一步设计引物PCR扩增目的片段，通过同源重组获得基因缺陷型菌株，使荚膜不能正常形成，暴露出细菌表面的抗原，既实现减毒的目的，又有获得广谱的减毒活苗或灭活苗的可能。
材料
细菌基因组DNA提取试剂盒、PCR琼脂糖凝胶回收试剂盒、质粒DNA小量提取试剂盒、购自TIANGEN，蛋白胨、酵母提取物购自OXIOD公司(英国)，PCR用rTaq酶、dNTPs、Buffer、MgCl 2购自大连宝生物技术公司，感受态刺激肽(CSP 1)购于南京金斯瑞。
实施例1
1.18CΔgalU的制备
1.1.设计引物
以肺炎链球菌18C(CCUG 7206)基因组DNA为模板，以NCBI上序列(编号WP_000202229.1)，使用primer premier5.0设计引物，引物由武汉金开瑞合成。
galU上游同源片段引物(UP)
P1(SEQ ID NO：1)：5’GTTGAAACTGCTGGTGCTCTTAA3’
P2(SEQ ID NO：2)：5’ATCAAACAAATTTTGGGCCCGG‐TCCGTGATAAATAACTTGGTAA 3’
galU下游同源片段引物(down)
P3(SEQ ID NO：3)：5’TCGTTAAGGGATCAACTTTGGGA‐TTTTCTTTCAACTTCGTCACAT3’
P4(SEQ ID NO：4)：5’TGCTTTCACTTTATTATCTTGG3’
红霉素抗性基因引物(Erm)
P5(SEQ ID NO：5)：5’ATGYGACGAAGAAGTTGAAAGAAAA3’
P6(SEQ ID NO：6)：5’TTACCAAGTTATTTATCACGGA3’
引物P2和P3分别带有22～23个分别与Erm基因5’和3’端两侧互补的碱基，这样扩增出的上下游同源片段分别带有一段与Erm基因互补的序列。
1.2.连接PCR扩增目的基因及胶回收
Up片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000001
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Down片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000002
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Erm片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000003
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收以上3个PCR产物，送武汉金开瑞测序验证。
连接片段PCR扩增体系和条件：
其中引物与模板的比例为1:50，3个PCR回收片段以等摩尔比例混匀后作为模板。
Figure PCTCN2019076919-appb-000004
Figure PCTCN2019076919-appb-000005
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收预期大小的PCR产物，送武汉金开瑞测序验证。
1.3.转化感受态的野生菌
将肺炎链球菌18C型培养于C+Y培养基至A550约0.1，加入感受态刺激因子CSP(100ng/ml)10μL，同时加入胶回收的连接PCR产物(100ng/ml)10μL，于37℃水浴90min，铺板于含红霉素0.25mg/L的TSA平板上，37℃培养一至两天至长出单菌落。
1.4 ΔgalU缺陷菌的PCR验证
挑取单个菌落培养，并提取基因组，PCR鉴定插入片段，胶回收后测序验证。验证正确，即为缺陷菌。将验证正确的单菌落在C+Y培养基中培养，当细菌密度达到A550约0.2左右时，加入甘油，混匀后保存于‐80℃冰箱。
2.缺陷菌毒力检测
2.1小鼠毒力实验(半数致死时间)
将雌性Balb/c小鼠随机分成两组，一组12只，一组13只。将肺炎链球菌野生型18C与galU缺陷株用PBS稀释至5×10 8CFU/ml，将100μL注射到小鼠腹腔，观察小鼠存活状态，记录小鼠死亡时间，并采用曼‐惠特尼U检验进行结果分析。实验结果：野生菌的半数致死时间为小时，而缺陷菌株的半数死亡时间为天，表明galU缺陷株的毒力显著下降。
2.2 TEM观察荚膜形态(野生菌荚膜厚而致密，缺陷菌荚膜薄而稀疏)
电镜前准备：C+Y中培养18CΔgalU缺陷菌，18C野生菌至A620约0.4，5000g，10min离心收集细菌，弃上清后立即沿壁加入戊二醛固定液1ml。电镜下可以看到：与18C野生菌比，缺陷菌的荚膜明显变薄变稀疏。
3.ΔgalU缺陷菌的主动免疫保护实验
3.1材料
23价多糖疫苗购于成都生物制品研究所，13价结合疫苗购于公司，CT佐剂购于SIGMA公司，铝佐剂购于Thermo公司，HRP标记羊抗人IgG购于武汉博士德，HRP标记羊抗人IgA购于武汉博士德。
3.2免疫策略
将Balb/c小鼠随机分为四大组，第一大组阳性对照PC(positivecontrol)为23价多糖疫苗和13价结合疫苗共3小组，每组10只；第二大组阴性对照NC(negative control)为CT佐剂加PBS共5小组，每组12只；第三大组皮下免疫(subcutaneous)组为18CΔgalU+铝佐剂共5小组，每组12只；第四大组鼻内免疫IN(intranasal)组为18CΔgalU+CT佐剂共5小组，每组12只。
首次免疫时，PC组2组腹腔注射0.1ml的23价多糖疫苗，1组腹腔注射0.1ml的13价结合疫苗；NC组5组都滴鼻30μl CT(1μg)+PBS；SC组5组都皮下注射100μl 10 8cfu18CΔgalU+100μl铝佐剂；IN组5组都滴鼻30μl cfu 18CΔgalU+CT(1μg)。两周后进行第二次免疫(PC组除外，只免疫一次)，NC组、SC组和IN组的免疫方法同首次免疫。四周后进行第三次加强免疫，此次免疫无需佐剂，免疫剂量如下：NC组30μl PBS；SC组200μl 10 8cfu18CΔgalU；IN组30μl 10 8cfu 18CΔgalU。
3.3 ELISA检测抗体效价
末次免疫一周后采集小鼠尾静脉血和唾液，其中唾液是用卡米可林30μl腹腔注射促进其分泌。分离血清，把唾液蛋白水平用PBS调到同水平，适当稀释后ELISA检测其抗体效价， 具体步骤如下：
1、包被：18CΔgalU菌株于C+Y中培养至A620约0.4左右，12000g，1min离心，PBS洗三次，抗原包被液重悬后稀释至A620为0.1，每孔包被100μl，4℃过夜，洗三次。
2、封闭：封闭液为2％BSA溶于PBST(0.1％Tween‐20)，300μl封闭2h，洗三次。
3、抗体：血清按1:100、1:200、1:400、……倍比稀释；唾液按1:25、1:50、1:100、……倍比稀释。每孔100μl，37℃45min，洗6次。
4、二抗：二抗按1:5000稀释，每孔100μl，37℃孵育15min。每孔加入50μl终止液显色，在450nm波长处测吸光度值(A450)。
抗体滴度定义为：实验组和阴性对照组光吸收比值大于或等于2.1时最大的血清稀释倍数。
3.4攻毒实验(败血症模型、肺炎模型)
末次免疫两周后进行攻毒实验，攻毒菌株选用野生型18C和国内流行的19A型。用18C有两种模型，败血症模型用3LD50的18C腹腔攻毒，肺炎模型选用1×10 8CFU的18C滴鼻攻毒；19A型用1×10 8CFU滴鼻攻毒。连续21天观察小鼠的生存状态，计算小鼠的生存率。
3.5结果统计
表1各组IgG与IgA抗体平均效价统计表
Figure PCTCN2019076919-appb-000006
从表1和附图1中可见，SC组血中IgG抗体效价虽然比IN组高，但唾液中并不存在sIgA，即并不诱导粘膜免疫反应，而IN组即可诱导粘膜免疫又可诱导系统免疫；SC组、IN组与NC组比效价明显高(p<0.01)。附图2说明接受我们减毒活菌18CΔgalU免疫的SC组和IN组小鼠的生存率显著高于阴性对照组NC组(p<0.01)，其中IN组的21日生存率可以达到100％，SC组13日前存活率可达100％，13日起下降至90％，21日平均存活率75％以上，几乎同已上市的23价多糖疫苗(PC组)和13价结合疫苗保护效果相似，无统计学差别，NC组存活率在7日内降至0，说明实验菌活性正常。附图3说明本疫苗对国内流行菌株19A的保护效果，SC组和IN组小鼠的生存率显著高于阴性对照NC组，其中IN组的生存率可以达到100％，SC组7日内生存率达100％，10日生存率降至90％，21日平均生存率可以达到80％以上，PC组5日起生存率下降至90％，每3‐5日以10％的速度下降，21日存活率不足75％，说明IN组的生存率比PC组明显要高，即保护效果明显好于23价多糖肺炎链球菌疫苗，NC组存活率在7日内降至0，说明实验菌活性正常。以上结果证明，无荚膜多糖的减毒肺炎链球菌疫苗，在小鼠体内能产生充足的保护力，也能保护小鼠免受其他血清型肺炎链球菌的攻击，鼻内免疫途径产生的保护效果要好于目前上市的23价肺炎多糖疫苗。
实施例2
1.19AΔgalU的制备
1.1.设计引物
以肺炎链球菌19A(CCUG 35180)基因组DNA为模板，以NCBI上序列(GenBank:
LQQK01000089.1)，使用primer premier5.0设计引物，引物由武汉金开瑞合成。
galU上游同源片段引物(UP)
P1(SEQ ID NO：1)：5’GTTGAAACTGCTGGTGCTCTTAA3’
P2(SEQ ID NO：2)：5’ATCAAACAAATTTTGGGCCCGG‐TCCGTGATAAATAACTTGGTAA 3’
galU下游同源片段引物(down)
P3(SEQ ID NO：3)：5’TCGTTAAGGGATCAACTTTGGGA‐TTTTCTTTCAACTTCGTCACAT3’
P4(SEQ ID NO：4)：5’TGCTTTCACTTTATTATCTTGG3’
红霉素抗性基因引物(Erm)
P5(SEQ ID NO：5)：5’ATGYGACGAAGAAGTTGAAAGAAAA3’
P6(SEQ ID NO：6)：5’TTACCAAGTTATTTATCACGGA3’
引物P2和P3分别带有22～23个分别与Erm基因5’和3’端两侧互补的碱基，这样扩增出的上下游同源片段分别带有一段与Erm基因互补的序列。
1.2.连接PCR扩增目的基因及胶回收
Up片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000007
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Down片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000008
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Erm片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000009
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收以上3个PCR产物，送武汉金开瑞测序验证。
连接片段PCR扩增体系和条件：
其中引物与模板的比例为1:50，3个PCR回收片段以等摩尔比例混匀后作为模板。
Figure PCTCN2019076919-appb-000010
Figure PCTCN2019076919-appb-000011
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收预期大小的PCR产物，送武汉金开瑞测序验证。
1.3.转化感受态的野生菌
将肺炎链球菌19A型培养于C+Y培养基至A550约0.1，加入感受态刺激因子CSP(100ng/ml)10μL，同时加入胶回收的连接PCR产物(100ng/ml)10μL，于37℃水浴90min，铺板于含红霉素0.25mg/L的TSA平板上，37℃培养一至两天至长出单菌落。
1.4 ΔgalU缺陷菌的PCR验证
挑取单个菌落培养，并提取基因组，PCR鉴定插入片段，胶回收后测序验证。验证正确，即为缺陷菌。将验证正确的单菌落在C+Y培养基中培养，当细菌密度达到A550约0.2左右时，加入甘油，混匀后保存于‐80℃冰箱。
2.缺陷菌毒力检测
2.1小鼠毒力实验(半数致死时间)
将雌性Balb/c小鼠随机分成两组，一组12只，一组13只。将肺炎链球菌野生型19A与galU缺陷株用PBS稀释至5×10 8CFU/ml，将100μL注射到小鼠腹腔，观察小鼠存活状态，记录小鼠死亡时间，并采用曼‐惠特尼U检验进行结果分析。实验结果：野生菌的半数致死时间为小时，而缺陷菌株的半数死亡时间为天，表明galU缺陷株的毒力显著下降。
2.2 TEM观察荚膜形态(野生菌荚膜厚而致密，缺陷菌荚膜薄而稀疏)
电镜前准备：C+Y中培养19AΔgalU缺陷菌，19A野生菌至A620约0.4，5000g，10min离心收集细菌，弃上清后立即沿壁加入戊二醛固定液1ml。电镜下可以看到：与19A野生菌比，缺陷菌的荚膜明显变薄变稀疏。
3.ΔgalU缺陷菌的主动免疫保护实验
3.1材料
23价多糖疫苗购于成都生物制品研究所，13价结合疫苗购于公司，CT佐剂购于SIGMA公司，铝佐剂购于Thermo公司，HRP标记羊抗人IgG购于武汉博士德，HRP标记羊抗人IgA购于武汉博士德。
3.2免疫策略
将Balb/c小鼠随机分为四大组，第一大组阳性对照PC(positivecontrol)为23价多糖疫苗和13价结合疫苗共3小组，每组10只；第二大组阴性对照NC(negative control)为CT佐剂加PBS共5小组，每组12只；第三大组皮下免疫(subcutaneous)组为19AΔgalU+铝佐剂共5小组，每组12只；第四大组鼻内免疫IN(intranasal)组为19AΔgalU+CT佐剂共5小组，每组12只。
首次免疫时，PC组2组腹腔注射0.1ml的23价多糖疫苗，1组腹腔注射0.1ml的13价结合疫苗；NC组5组都滴鼻30μl CT(1μg)+PBS；SC组5组都皮下注射100μl 10 8cfu19AΔgalU+100μl铝佐剂；IN组5组都滴鼻30μl cfu 19AΔgalU+CT(1μg)。两周后进行第二次免疫(PC组除外，只免疫一次)，NC组、SC组和IN组的免疫方法同首次免疫。四周后进行第三次加强免疫，此次免疫无需佐剂，免疫剂量如下：NC组30μl PBS；SC组200μl 10 8cfu19AΔgalU；IN组30μl 10 8cfu 19AΔgalU。
3.3 ELISA检测抗体效价
末次免疫一周后采集小鼠尾静脉血和唾液，其中唾液是用卡米可林30μl腹腔注射促进 其分泌。分离血清，把唾液蛋白水平用PBS调到同水平，适当稀释后ELISA检测其抗体效价，具体步骤如下：
1、包被：19AΔgalU菌株于C+Y中培养至A620约0.4左右，12000g，1min离心，PBS洗三次，抗原包被液重悬后稀释至A620为0.1，每孔包被100μl，4℃过夜，洗三次。
2、封闭：封闭液为2％BSA溶于PBST(0.1％Tween‐20)，300μl封闭2h，洗三次。
3、抗体：血清按1:100、1:200、1:400、……倍比稀释；唾液按1:25、1:50、1:100、……倍比稀释。每孔100μl，37℃45min，洗6次。
4、二抗：二抗按1:5000稀释，每孔100μl，37℃孵育15min。每孔加入50μl终止液显色，在450nm波长处测吸光度值(A450)。
抗体滴度定义为：实验组和阴性对照组光吸收比值大于或等于2.1时最大的血清稀释倍数。
3.4攻毒实验(败血症模型、肺炎模型)
末次免疫两周后进行攻毒实验，攻毒菌株选用野生型19A和国内流行的1型。用19A有两种模型，败血症模型用3LD50的19A腹腔攻毒，肺炎模型选用1×10 8CFU的19A滴鼻攻毒；1型用1×10 8CFU滴鼻攻毒。连续21天观察小鼠的生存状态，计算小鼠的生存率。
3.5结果统计
表2各组IgG与IgA抗体平均效价统计表
Figure PCTCN2019076919-appb-000012
从表2和附图4中可见，SC组血中IgG抗体效价虽然比IN组高，但唾液中并不存在sIgA，即并不诱导粘膜免疫反应，而IN组即可诱导粘膜免疫又可诱导系统免疫；SC组、IN组与NC组比效价明显高(p<0.01)。附图5说明接受我们减毒活菌19AΔgalU免疫的SC组合IN组小鼠的生存率显著高于阴性对照组NC组(p<0.01)，其中IN组的21日生存率可以达到100％，SC组7日存活率达100％，9日起下降至90％，21日平均存活率75％以上，几乎同已上市的23价多糖疫苗(PC组)和13价结合疫苗保护效果相似，无统计学差别，NC组存活率在7日内降至0，说明实验菌活性正常。附图6说明本疫苗对国内流行菌株1型的保护效果，SC组和IN组小鼠的生存率显著高于阴性对照NC组，其中IN组的21日生存率可达100％，SC组7日生存率可达100％，9日起下降至90％，21日平均生存率可达80％以上，PC组5日起生存率下降至90％，每3‐5日以10％的速度下降，21日存活率不足75％，说明IN组的生存率比PC组明显要高，即保护效果明显好于23价多糖肺炎链球菌疫苗，NC组存活率在7日内降至0，说明实验菌活性正常。以上结果证明，无荚膜多糖的减毒肺炎链球菌疫苗，在小鼠体内能产生充足的保护力，也能保护小鼠免受其他血清型肺炎链球菌的攻击，鼻内免疫途径产生的保护效果要好于目前上市的23价肺炎多糖疫苗。
实施例3
1.9VΔgalU的制备
1.1.设计引物
以肺炎链球菌9V(CCUG 36618)基因组DNA为模板，以NCBI上序列(编号NZ_MAVR01000057.1)，使用primer premier5.0设计引物，引物由武汉金开瑞合成。
galU上游同源片段引物(UP)
P1(SEQ ID NO：1)：5’GTTGAAACTGCTGGTGCTCTTAA3’
P2(SEQ ID NO：2)：5’ATCAAACAAATTTTGGGCCCGG‐TCCGTGATAAATAACTTGGTAA 3’
galU下游同源片段引物(down)
P3(SEQ ID NO：3)：5’TCGTTAAGGGATCAACTTTGGGA‐TTTTCTTTCAACTTCGTCACAT3’
P4(SEQ ID NO：4)：5’TGCTTTCACTTTATTATCTTGG3’
红霉素抗性基因引物(Erm)
P5(SEQ ID NO：5)：5’ATGYGACGAAGAAGTTGAAAGAAAA3’
P6(SEQ ID NO：6)：5’TTACCAAGTTATTTATCACGGA3’
引物P2和P3分别带有22～23个分别与Erm基因5’和3’端两侧互补的碱基，这样扩增出的上下游同源片段分别带有一段与Erm基因互补的序列。
1.2.连接PCR扩增目的基因及胶回收
Up片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000013
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Down片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000014
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Erm片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000015
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收以上3个PCR产物，送武汉金开瑞测序验证。
连接片段PCR扩增体系和条件：
其中引物与模板的比例为1:50，3个PCR回收片段以等摩尔比例混匀后作为模板。
Figure PCTCN2019076919-appb-000016
Figure PCTCN2019076919-appb-000017
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收预期大小的PCR产物，送武汉金开瑞测序验证。
1.3.转化感受态的野生菌
将肺炎链球菌9V型培养于C+Y培养基至A550约0.1，加入感受态刺激因子CSP(100ng/ml)10μL，同时加入胶回收的连接PCR产物(100ng/ml)10μL，于37℃水浴90min，铺板于含红霉素0.25mg/L的TSA平板上，37℃培养一至两天至长出单菌落。
1.4 ΔgalU缺陷菌的PCR验证
挑取单个菌落培养，并提取基因组，PCR鉴定插入片段，胶回收后测序验证。验证正确，即为缺陷菌。将验证正确的单菌落在C+Y培养基中培养，当细菌密度达到A550约0.2左右时，加入甘油，混匀后保存于‐80℃冰箱。
2.缺陷菌毒力检测
2.1小鼠毒力实验(半数致死时间)
将雌性Balb/c小鼠随机分成两组，一组12只，一组13只。将肺炎链球菌野生型9V与galU缺陷株用PBS稀释至5×10 8CFU/ml，将100μL注射到小鼠腹腔，观察小鼠存活状态，记录小鼠死亡时间，并采用曼‐惠特尼U检验进行结果分析。实验结果：野生菌的半数致死时间为小时，而缺陷菌株的半数死亡时间为天，表明galU缺陷株的毒力显著下降。
2.2 TEM观察荚膜形态(野生菌荚膜厚而致密，缺陷菌荚膜薄而稀疏)
电镜前准备：C+Y中培养9VΔgalU缺陷菌，9V野生菌至A620约0.4，5000g，10min离心收集细菌，弃上清后立即沿壁加入戊二醛固定液1ml。电镜下可以看到：与9V野生菌比，缺陷菌的荚膜明显变薄变稀疏。
3.ΔgalU缺陷菌的主动免疫保护实验
3.1材料
23价多糖疫苗购于成都生物制品研究所，13价结合疫苗购于公司，CT佐剂购于SIGMA公司，铝佐剂购于Thermo公司，HRP标记羊抗人IgG购于武汉博士德，HRP标记羊抗人IgA购于武汉博士德。
3.2免疫策略
将Balb/c小鼠随机分为四大组，第一大组阳性对照PC(positivecontrol)为23价多糖疫苗和13价结合疫苗共3小组，每组10只；第二大组阴性对照NC(negative control)为CT佐剂加PBS共5小组，每组12只；第三大组皮下免疫(subcutaneous)组为9VΔgalU+铝佐剂共5小组，每组12只；第四大组鼻内免疫IN(intranasal)组为9VΔgalU+CT佐剂共5小组，每组12只。
首次免疫时，PC组2组腹腔注射0.1ml的23价多糖疫苗，1组腹腔注射0.1ml的13价结合疫苗；NC组5组都滴鼻30μl CT(1μg)+PBS；SC组5组都皮下注射100μl 10 8cfu9VΔgalU+100μl铝佐剂；IN组5组都滴鼻30μl cfu 9VΔgalU+CT(1μg)。两周后进行第二次免疫(PC组除外，只免疫一次)，NC组、SC组和IN组的免疫方法同首次免疫。四周后进行第三次加强免疫，此次免疫无需佐剂，免疫剂量如下：NC组30μl PBS；SC组200μl 10 8cfu9VΔgalU；IN组30μl 10 8cfu 9VΔgalU。
3.3 ELISA检测抗体效价
末次免疫一周后采集小鼠尾静脉血和唾液，其中唾液是用卡米可林30μl腹腔注射促进 其分泌。分离血清，把唾液蛋白水平用PBS调到同水平，适当稀释后ELISA检测其抗体效价，具体步骤如下：
1、包被：9VΔgalU菌株于C+Y中培养至A620约0.4左右，12000g，1min离心，PBS洗三次，抗原包被液重悬后稀释至A620为0.1，每孔包被100μl，4℃过夜，洗三次。
2、封闭：封闭液为2％BSA溶于PBST(0.1％Tween‐20)，300μl封闭2h，洗三次。
3、抗体：血清按1:100、1:200、1:400、……倍比稀释；唾液按1:25、1:50、1:100、……倍比稀释。每孔100μl，37℃45min，洗6次。
4、二抗：二抗按1:5000稀释，每孔100μl，37℃孵育15min。每孔加入50μl终止液显色，在450nm波长处测吸光度值(A450)。
抗体滴度定义为：实验组和阴性对照组光吸收比值大于或等于2.1时最大的血清稀释倍数。
3.4攻毒实验(败血症模型、肺炎模型)
末次免疫两周后进行攻毒实验，攻毒菌株选用野生型9V和国内流行的23F型。用9V有两种模型，败血症模型用3LD50的9V腹腔攻毒，肺炎模型选用1×10 8CFU的9V滴鼻攻毒；23F型用1×10 8CFU滴鼻攻毒。连续21天观察小鼠的生存状态，计算小鼠的生存率。
3.5结果统计
表3各组IgG与IgA抗体平均效价统计表
Figure PCTCN2019076919-appb-000018
从表3和附图7中可见，SC组血中IgG抗体效价虽然比IN组高，但唾液中并不存在sIgA，即并不诱导粘膜免疫反应，而IN组即可诱导粘膜免疫又可诱导系统免疫；SC组、IN组与NC组比效价明显高(p<0.01)。附图8说明接受我们减毒活菌9VΔgalU免疫的SC组合IN组小鼠的生存率显著高于阴性对照组NC组(p<0.01)，其中IN组的21日生存率可以达到100％，SC组7日存活率达100％，12日起下降至90％，21日平均存活率75％以上，几乎同已上市的23价多糖疫苗(PC组)和13价结合疫苗保护效果相似，无统计学差别，NC组存活率在7日内降至0，说明实验菌活性正常。附图9说明本疫苗对国内流行菌株23F型的保护效果，SC组和IN组小鼠的生存率显著高于阴性对照NC组，其中IN组的21日生存率可达100％，SC组7日生存率可达100％，10日起下降至90％，21日平均生存率可达80％以上，PC组5日起生存率下降至90％，每3‐5日以10％的速度下降，21日存活率不足75％，说明IN组的生存率比PC组明显要高，即保护效果明显好于23价多糖肺炎链球菌疫苗，NC组存活率在7日内降至0，说明实验菌活性正常。以上结果证明，无荚膜多糖的减毒肺炎链球菌疫苗，在小鼠体内能产生充足的保护力，也能保护小鼠免受其他血清型肺炎链球菌的攻击，鼻内免疫途径产生的保护效果要好于目前上市的23价肺炎多糖疫苗。
实施例4
1.3ΔgalU的制备
1.1.设计引物
以肺炎链球菌3型(CCUG 6798)基因组DNA为模板，以NCBI上序列(编号NZ_LSLM01000003.1)，使用primer premier5.0设计引物，引物由武汉金开瑞合成。
galU上游同源片段引物(UP)
P1(SEQ ID NO：1)：5’GTTGAAACTGCTGGTGCTCTTAA3’
P2(SEQ ID NO：2)：5’ATCAAACAAATTTTGGGCCCGG‐TCCGTGATAAATAACTTGGTAA 3’
galU下游同源片段引物(down)
P3(SEQ ID NO：3)：5’TCGTTAAGGGATCAACTTTGGGA‐TTTTCTTTCAACTTCGTCACAT3’
P4(SEQ ID NO：4)：5’TGCTTTCACTTTATTATCTTGG3’
红霉素抗性基因引物(Erm)
P5(SEQ ID NO：5)：5’ATGYGACGAAGAAGTTGAAAGAAAA3’
P6(SEQ ID NO：6)：5’TTACCAAGTTATTTATCACGGA3’
引物P2和P3分别带有22～23个分别与erm基因5’和3’端两侧互补的碱基，这样扩增出的上下游同源片段分别带有一段与erm基因互补的序列。
1.2.连接PCR扩增目的基因及胶回收
Up片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000019
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Down片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000020
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
Erm片段的扩增体系和条件：
Figure PCTCN2019076919-appb-000021
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收以上3个PCR产物，送武汉金开瑞测序验证。
连接片段PCR扩增体系和条件：
其中引物与模板的比例为1:50，3个PCR回收片段以等摩尔比例混匀后作为模板。
Figure PCTCN2019076919-appb-000022
Figure PCTCN2019076919-appb-000023
PCR反应条件：95℃预变性5min，1cycle；95℃变性1min，55℃退火30s，72℃延伸1min，30cycle；72℃延伸10min，1cycle。
凝胶回收试剂盒回收预期大小的PCR产物，送武汉金开瑞测序验证。
1.3.转化感受态的野生菌
将肺炎链球菌3型培养于C+Y培养基至A550约0.1，加入感受态刺激因子CSP(100ng/ml)10μL，同时加入胶回收的连接PCR产物(100ng/ml)10μL，于37℃水浴90min，铺板于含红霉素0.25mg/L的TSA平板上，37℃培养一至两天至长出单菌落。
1.4 ΔgalU缺陷菌的PCR验证
挑取单个菌落培养，并提取基因组，PCR鉴定插入片段，胶回收后测序验证。验证正确，即为缺陷菌。将验证正确的单菌落在C+Y培养基中培养，当细菌密度达到A550约0.2左右时，加入甘油，混匀后保存于‐80℃冰箱。
2.缺陷菌毒力检测
2.1小鼠毒力实验(半数致死时间)
将雌性Balb/c小鼠随机分成两组，一组12只，一组13只。将肺炎链球菌野生型3与galU缺陷株用PBS稀释至5×10 8CFU/ml，将100μL注射到小鼠腹腔，观察小鼠存活状态，记录小鼠死亡时间，并采用曼‐惠特尼U检验进行结果分析。实验结果：野生菌的半数致死时间为小时，而缺陷菌株的半数死亡时间为天，表明galU缺陷株的毒力显著下降。
2.2 TEM观察荚膜形态(野生菌荚膜厚而致密，缺陷菌荚膜薄而稀疏)
电镜前准备：C+Y中培养3ΔgalU缺陷菌，3型野生菌至A620约0.4，5000g，10min离心收集细菌，弃上清后立即沿壁加入戊二醛固定液1ml。电镜下可以看到：与3型野生菌比，缺陷菌的荚膜明显变薄变稀疏。
3.ΔgalU缺陷菌的主动免疫保护实验
3.1材料
23价多糖疫苗购于成都生物制品研究所，13价结合疫苗购于公司，CT佐剂购于SIGMA公司，铝佐剂购于Thermo公司，HRP标记羊抗人IgG购于武汉博士德，HRP标记羊抗人IgA购于武汉博士德。
3.2免疫策略
将Balb/c小鼠随机分为四大组，第一大组阳性对照PC(positivecontrol)为23价多糖疫苗和13价结合疫苗共3小组，每组10只；第二大组阴性对照NC(negative control)为CT佐剂加PBS共5小组，每组12只；第三大组皮下免疫(subcutaneous)组为3ΔgalU+铝佐剂共5小组，每组12只；第四大组鼻内免疫IN(intranasal)组为3ΔgalU+CT佐剂共5小组，每组12只。
首次免疫时，PC组2组腹腔注射0.1ml的23价多糖疫苗，1组腹腔注射0.1ml的13价结合疫苗；NC组5组都滴鼻30μl CT(1μg)+PBS；SC组5组都皮下注射100μl 10 8cfu3ΔgalU+100μl铝佐剂；IN组5组都滴鼻30μl cfu 3ΔgalU+CT(1μg)。两周后进行第二次免疫(PC组除外，只免疫一次)，NC组、SC组和IN组的免疫方法同首次免疫。四周后进行第三次加强免疫，此次免疫无需佐剂，免疫剂量如下：NC组30μl PBS；SC组200μl 10 8cfu 3ΔgalU；IN组30μl 10 8cfu 3ΔgalU。
3.3 ELISA检测抗体效价
末次免疫一周后采集小鼠尾静脉血和唾液，其中唾液是用卡米可林30μl腹腔注射促进 其分泌。分离血清，把唾液蛋白水平用PBS调到同水平，适当稀释后ELISA检测其抗体效价，具体步骤如下：
1、包被：3ΔgalU菌株于C+Y中培养至A620约0.4左右，12000g，1min离心，PBS洗三次，抗原包被液重悬后稀释至A620为0.1，每孔包被100μl，4℃过夜，洗三次。
2、封闭：封闭液为2％BSA溶于PBST(0.1％Tween‐20)，300μl封闭2h，洗三次。
3、抗体：血清按1:100、1:200、1:400、……倍比稀释；唾液按1:25、1:50、1:100、……倍比稀释。每孔100μl，37℃45min，洗6次。
4、二抗：二抗按1:5000稀释，每孔100μl，37℃孵育15min。每孔加入50μl终止液显色，在450nm波长处测吸光度值(A450)。
抗体滴度定义为：实验组和阴性对照组光吸收比值大于或等于2.1时最大的血清稀释倍数。
3.4攻毒实验(败血症模型、肺炎模型)
末次免疫两周后进行攻毒实验，攻毒菌株选用野生型3型和国内流行的14型。用3型有两种模型，败血症模型用3LD50的3型腹腔攻毒，肺炎模型选用1×10 8CFU的3型滴鼻攻毒；14型用1×10 8CFU滴鼻攻毒。连续21天观察小鼠的生存状态，计算小鼠的生存率。
3.5结果统计
表4各组IgG与IgA抗体平均效价统计表
Figure PCTCN2019076919-appb-000024
从表4和附图10中可见，SC组血中IgG抗体效价虽然比IN组高，但唾液中并不存在sIgA，即并不诱导粘膜免疫反应，而IN组即可诱导粘膜免疫又可诱导系统免疫；SC组、IN组与NC组比效价明显高(p<0.01)。附图11说明接受我们减毒活菌3ΔgalU免疫的SC组和IN组小鼠的生存率显著高于阴性对照组NC组(p<0.01)，其中IN组的21日生存率可以达到100％，SC组7日存活率达100％，9日起下降至90％，21日平均存活率75％以上，几乎同已上市的23价多糖疫苗(PC组)和13价结合疫苗保护效果相似，无统计学差别，NC组存活率在7日内降至0，说明实验菌活性正常。附图12说明本疫苗对国内流行菌株14型的保护效果，SC组和IN组小鼠的生存率显著高于阴性对照NC组，其中IN组的21日生存率可达100％，SC组7日生存率可达100％，8日起下降至90％，21日平均生存率可达80％以上，PC组5日起生存率下降至90％，每3‐5日以10％的速度下降，21日存活率不足75％，说明IN组的生存率比PC组明显要高，即保护效果明显好于23价多糖肺炎链球菌疫苗，NC组存活率在7日内降至0，说明实验菌活性正常。以上结果证明，无荚膜多糖的减毒肺炎链球菌疫苗，在小鼠体内能产生充足的保护力，也能保护小鼠免受其他血清型肺炎链球菌的攻击，鼻内免疫途径产生的保护效果要好于目前上市的23价肺炎多糖疫苗。
最后有必要在此说明的是：以上实施例只用于对本发明的技术方案作进一步详细地说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。
Figure PCTCN2019076919-appb-000025
Figure PCTCN2019076919-appb-000026

Claims (10)

  1. 一种肺炎链球菌疫苗，其特征在于：所述肺炎链球菌疫苗包括至少一个血清型的肺炎链球菌为抗原，所述肺炎链球菌为经敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因后的无荚膜多糖肺炎链球菌。
  2. 根据权利要求1所述的肺炎链球菌疫苗，其特征在于，所述肺炎链球菌包括但不限于以下血清型中的一种或以上的组合：1、2、3、4、5、6A、6B、6C、7A、7B、7C、7F、8、9A、9L、9N、9V、10A、10B、10C、10F、11A、11B、11C、11D、11F、12A、12B、12F、13、14、15A、15B、15C、15F、16A、16F、17A、17F、18A、18B、18C、18F、19A、19B、19C、19F、20、21、22A、22F、23A、23B、23F、24A、24B、24F、25A、25F、27、28A、28F、29、31、32A、32F、33A、33B、33C、33D、33F、34、35A、35B、35C、36、37、38、39、40、41、41F、42、43、44、45、46、47A、47F和/或48。
  3. 根据权利要求1所述的肺炎链球菌疫苗，其特征在于，所述基因选自以下基因中的一种或以上的组合：cps基因、galU、CcpA和/或PGM。
  4. 根据权利要求5所述的肺炎链球菌疫苗，其特征在于，cps基因选自以下基因中的一种或以上的组合：cpsA、cpsB、cpsC、cpsD、cpsE、CpsF、CpsG和/或CpsI。
  5. 一种无荚膜肺炎链球菌，其特征在于：所述肺炎链球菌为敲除荚膜多糖合成途径和/或影响荚膜多糖表达相关基因的肺炎链球菌。
  6. 一种无荚膜糖肺炎链球菌的制备方法，其特征在于：所述方法采用基因敲除的方法敲除影响肺炎链球菌荚膜多糖合成的基因cps、galU、CcpA和/或PGM。
  7. 根据权利要求7所述的无荚膜糖肺炎链球菌的制备方法，其特征在于，所述基因的敲除方法具体为：
    a)设计引物：根据影响肺炎链球菌荚膜多糖合成基因的上下游位点，设计引物；
    b)PCR：采用步骤a)中的引物扩增基因片段，胶回收对应片段；
    c)转化感受态：培养野生菌后加入感受态刺激因子及胶回收的片段，培养至单菌落；
    d)选取缺陷菌：挑取步骤c)中长出的单菌落进行扩大培养，获得无影响肺炎链球菌荚膜多糖合成基因的缺陷菌。
  8. 一种肺炎链球菌疫苗的制备方法，其特征在于，所述方法以权利要求1～3任一项所述的至少一种血清型的肺炎链球菌经敲除至少一个荚膜基因后，转化筛选得到无 荚膜肺炎链球菌疫苗。
  9. 一种无荚膜糖肺炎链球菌的制备方法，其特征在于：所述方法采用基因敲除的方法敲除影响肺炎链球菌荚膜多糖合成的基因galU。
  10. 一种肺炎链球菌抗原，其特征在于：所述肺炎链球菌抗原为经敲除或修饰影响荚膜多糖合成途径和/或影响荚膜多糖表达相关基因后的无荚膜多糖肺炎链球菌。
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