Preparation method of pseudomonas protegens transcription factor LexA polyclonal antibody

[0025]Example 1

[0026]A method for preparing a modified fake skeleton transcription factor Lexa polyclonal antibody in this example, wherein the specific embodiment of the recombinant plasmid is constructed is as follows: Deformation pseudocensis Lexa gene sequence obtained from the previous genome sequence, manually synthesized Lexa gene And insert it into a PET-32 expression vector, inserted into a gene nucleic acid sequence such asfigure 1 SEQ ID NO: 1 showed; PET-32 vector is ampic resistance, containing TRX tags and 6xHIS tags. The TRX sulfur oxygen label facilitates reinforcing protein soluble expression, the molecular weight of approximately 20 kDa, located in the N-end of the target gene in the PET-32 vector. The constructed recombinant plasmid was converted into E. coli DH5α, using LB plate containing 100 μg / ml ampicillin, 37 ° C for 12 hours, and the filamental rock, extract plasmid and sequencing, sequencing, the desired recombination Plasmid.

[0027]Example 2

[0028]A method of preparing a modified homophilic antibody in the present example, wherein the specific embodiment of the sample expression is as follows: the constructing recombinant plasmids described in Example 1 transformed BL21 DE3 sensitive state cells, inoculation resistance LB Tablet medium, growing 24 hours; 6 monoclons of transformed plates were selected, and 3 ml of resistant liquid medium was seeded; 37 ° C, 220 rpm was cultured to OD600NM0.5-0.6, and 0.5 mM IPTG was induced induced by 3.5 hours; centrifugation The bacteria, ultrasonic crushing, and SDS-PAGE detection expression. The test results show that the protein is expressed in the upper part of the liquid and the inclusion body, and soluble expression is purified.

[0029]Example 3

[0030]The method of preparing a variant homophilic antibody in the present example, wherein the specific embodiment of the specific expression is as follows: Selecting the small species of a small expression of a small expression of a small expression in which 60 μL to 200 ml of resistant medium, 37 ° C, cultured for 24 hours; fresh resistance medium to 800 ml, incubation for 1-2 hours, to OD600NM0.5-0.6; 200 μL of 1M IPTG (28 ° C or 37 ° C) induced expression of 3.5 hours; 4 ° C centrifugation The bacteria (66 rpm), precipitate and discarded the upper cleaner, add 30 mlpbst suspension, add 1 mm PMSF, and the ultrasonic wave 200W under ice bath is 6 min; shake the bed at 4 ° C for 1 hour; 133 rpm The second high speed is centrifuged for 15 minutes, take the upper part to the 400 μl of nickel column 4 ° C for 24 hours; collect the nickel column (33 rpm), 20 mm imidazole wash solution to wash the beads, wash it off the miscellaneous protein (1 ml × 3 times) Add 300 μL of 300 mm imidazole eluent, allow the eluate to be fully combined with Beads for 1 hour, centrifugally collected the upper liquid; add 300 μl of eluent again to beads, eluent for 1 hour, centrifuge, and collected from the upper part. Two eluents were combined with a test tube; use PBS dialysis; SDS-PAGE identified protein molecular weight, purity and concentration. Detection resultsimage 3 As shown, the target protein is a soluble upper liquid expression, with a total amount of 3 mg, and the molecular weight is 42 kDa.