Streptococcus protective antigen C5a and preparation method thereof
A protective antigen, streptococcus technology, applied in bacterial antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of SEZ difficulty and achieve good immune response effect
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Embodiment 1
[0027] Embodiment one, bacterial strain and growth condition
[0028] The bacterial strain adopts SEZ C55138 strain; the medium is TSB medium or TSA medium containing 5% fetal bovine serum; shake culture at 37° C. for about 8 hours, or the OD600 reaches 0.6-1.0.
[0029] The carrier is Escherichia coli pET-28a, and the competent cell is Escherichia coli BL21.
[0030] SEZ C55138 strain, Escherichia coli pET-28a vector and Escherichia coli BL21 competent cells are all commercial products.
Embodiment 2
[0031] Embodiment two, preparation method
[0032] The SCPZ protein is encoded by the scpZ gene, the forward primer of the gapdh gene has a BamHI restriction site, and the reverse primer has an EcoRI restriction site. The forward and reverse primers were designed from the genome sequence of SEZ MGCS10565 (NCBI Accession: CP001129.1).
[0033] The preparation method of Streptococcus equi subspecies zooepidemic protective antigen GAPDH comprises the following steps:
[0034] PCR amplification: use the genome of the C55138 strain as a template, and use the following primers for PCR amplification;
[0035] Forward primer (SEQ ID NO.3): 5'-CAGAGGGC GGATCC TTT-3', the underlined part is the restriction site of BamHI,
[0036] Reverse primer (SEQ ID NO.4): 5'-CTGCTG GAATTC TGAGATAT-3', the underlined part is the EcoRI restriction site;
[0037] Ligation with the vector: the PCR product was digested with a restriction enzyme and then ligated with the pET-32a vector digested with...
Embodiment 3
[0046] Embodiment 3. Mice immunization and challenge test
[0047] 1. Thirty 4-week-old BALB / c female mice were randomly divided into 3 groups, 10 in each group;
[0048] 2. Experimental group: After 50 μg of purified recombinant SCPZ protein was emulsified with Freund’s complete adjuvant, the mice in group 1 were immunized by intraperitoneal injection. After 14 days, the same antigen emulsified with 50 μg of Freund’s incomplete adjuvant was used in the same way. Secondary injection of immunized mice;
[0049] 3. Positive control group: Inject SEZ inactivated vaccine (use SEZ C55138 strain inactivated by Freund's complete adjuvant and Freund's incomplete adjuvant to emulsify and inactivate the mice in the second group, the bacterial concentration is 2×10 8 CFU / mL);
[0050] 4. Negative control group: inject PBS emulsified with adjuvant to the mice of the third group, and the adjuvant used is the same as that of the positive control group;
[0051] 5. Ten days after all mice...
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