Probiotic medicine or health care product for treating diseases
A technology of probiotics and uses, which is applied in the field of probiotic drugs or health products to achieve the effect of promoting therapeutic effects and good application prospects
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Embodiment 1
[0032] Example 1 Preparation of MAdCAM-1
[0033] Design primers, F: 5'-TGGATCCATGCAAAGCTTGCAGGTGAAG-3', R: 5'-AAGGATCCTTAGACGGGGATGGCCTGG-3', using human DNA as a template, using F and R as primers, using PCR to amplify at 94°C for 1 min, 49°C 30s, 72°C for 1min)×5; (94°C for 1min, 65°C for 30s, 72°C for 1min)×25, a total of 30 cycles. Recover the PCR product and clone it into the pGEM-T vector. After the identification is correct, double enzyme digestion to obtain MAdCAM-1 is connected to the pQE30 vector. After the successful recombination of the plasmid, after double enzyme digestion, electrophoresis can produce two bands, which are Digestion product of MAdCAM-1 insert and PQE30 vector. The positive clones were sent to Invitrogen Company for sequencing, and the sequencing results were consistent with the sequence of MAdCAM-1 in GenBank.
[0034] The pQE30-MAdCAM-1 recombinant plasmid was transformed into M15 competent cells and inoculated in LB solid medium; after incuba...
Embodiment 2
[0036] Example 2 Preparation of MAdCAM-1 Monoclonal Antibody
[0037] 1) Immunization of BALB / c mice. ① The first immunization: Add 100 μL of CFA to 100 μL of 0.5 mg / mL recombinant MAdCAM-1 protein prepared in Example 1 for emulsification, and immunize 6-8 week-old mice with the above emulsion. ② Second immunization: 2 to 3 weeks after the first immunization, 100 μL of IFA was added to 100 μL of 0.5 mg / mL recombinant MAdCAM-1 protein for emulsification, and the above emulsion was used to immunize the above-mentioned 6 to 8-week-old mice for the second time. mice. ③The third immunization: the third immunization was carried out 2 to 3 weeks after the completion of the second immunization. The method is the same as the second immunization. On the 10th day after the completion of the third immunization, blood was collected from the tail vein. 2) Determination of antibody titer. Coat the 96-well microtiter plate with 1 μg / mL antigen solution; dilute 1:100 mouse serum in the 96...
Embodiment 34
[0041] Example 3 Characterization of 4C7 antibody
[0042] (1) Determination of monoclonal antibody affinity: Indirect ELISA method was used to coat the microtiter plate with MAdCAM-1 at a concentration of 1 μg / mL. Mouse IgG is the secondary antibody, and the OD450nm absorbance value is read by a microplate reader. When the OD450nm readings of several consecutive dilutions no longer increase, it is regarded as 100% combination of antigen and antibody. Take the antibody concentration (mol / L) as the abscissa, and the OD450nm absorbance value as the ordinate to make a scatter diagram, and take half of the maximum value of the reading The antigen-antibody binding rate was 50%, and a logarithmic trend line and formula were generated. Substitute half of the maximum value of OD450nm into the formula to obtain the antibody concentration at this time, which is the affinity dissociation constant (Kd). The results showed that the affinity relay constant of 4C7 monoclonal antibody was 2...
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