Hybridoma cell strain capable of secreting monoclonal antibody neutralizing clostridium septicum alpha toxin and application thereof
A hybridoma cell line and monoclonal antibody technology, applied in the biological field, to achieve the effect of strong specificity, high sensitivity, and easy sample operation
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Embodiment 1
[0038] Embodiment 1rCSA, rCSA m4△11 And the preparation of natural toxin of Clostridium putrefaciens
[0039] (1) rCSA and rCSA m4△11 expression and purification of
[0040] (11) According to the known gene sequence of Clostridium putrefaction alpha toxin, after codon optimization, a coding sequence of 6 histidine tags is added to the 3' end of the gene. The gene fragment Gcsa and the gene fragment Gcsa containing 4 amino acid mutations (C54L, N264A, H269A and W310A) and 11 amino acid (212th to 222nd) deletions were obtained by chemical synthesis. m4△11 .
[0041] The nucleotide sequence of the gene fragment Gcsa is shown in SEQ ID NO.1;
[0042] The gene fragment Gcsa m4△11 The nucleotide sequence of is shown in SEQ ID NO.2.
[0043] (12) gene fragment Gcsa and gene fragment Gcsa m4△11 Cloned into the prokaryotic expression vector pET-30a(+), respectively, to obtain the prokaryotic expression plasmids pET-Gcsa and pET-Gcsa m4△11 .
[0044] (13) Prokaryotic expression p...
Embodiment 2
[0050] Example 2 Screening of hybridoma cell lines secreting neutralizing Clostridium putrefaction alpha toxin monoclonal antibody hybridoma cell line and preparation of monoclonal antibody ascites
[0051] (1) Immunization and antibody titer determination of BALB / c mice
[0052] BALB / c mice aged 6-8 weeks were immunized with inactivated rCSA as the immunizing antigen, and immunized 4 times with an interval of 2 weeks between each time. For the first immunization, add an equal volume of Freund's complete adjuvant to the immune antigen, emulsify, and immunize BALB / c mice by subcutaneous injection at multiple points on the back of the neck, 50 μg per mouse; for the second and third immunizations, replace the complete Freund's adjuvant Freund's incomplete adjuvant was used, and the others were the same as the first immunization; 7 to 10 days after the third immunization, a small amount of blood was collected from the tail, and the purified rCSA m4△11 The serum titer was determin...
Embodiment 31F4
[0057] The in vitro neutralization test results of the ascites control (S) of embodiment 31F4, 3D8 and 1G6 and myeloma cell SP2 / 0
[0058] Carry out according to the serum neutralization method stipulated in "Veterinary Pharmacopoeia of the People's Republic of China" (2015 edition three).
[0059] Take 0.4mL of 1F4, 3D8 and 1G6 three kinds of ascites and mix them with 0.8mL of Clostridium putrefaciens natural toxin (containing different mouse MLD, such as 4, 8, 12, etc.) 2 20g mice, 0.3mL / mouse, at the same time, take 0.4mL of ascites control (S) and 0.8mL of Clostridium putrefaciens natural toxin (containing 4 mouse MLD) mixed as challenge control, and observe for 72h. The results showed that 2 / 2 of the mice in the challenge control group died within 72 hours, while the three ascites neutralizing activities of 1F4, 3D8 and 1G6 could reach 1, 2 and 1 mouse MLD / 0.1 mL, respectively. For this reason, 3D8 with the highest neutralizing activity was selected for subsequent experi...
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