A kind of anti-human nerve growth factor scfv antibody and its preparation method
A growth factor, human nerve technology, applied in the field of anti-human nerve growth factor scFv antibody and preparation, can solve the problems of low expression, complex glycosylation modification, etc., and achieve the effects of low cost, high binding ability and broad market prospect.
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Embodiment 1
[0035] Example 1: Acquisition and synthesis of anti-human nerve growth factor scFv antibody DNA sequence
[0036] Template: three existing anti-human nerve growth factor antibodies (parental antibody A: a human anti-NGF neutralizing antibody as a selective NGF pathway inhibitor, which is the anti-human nerve growth factor obtained in Example 2 of Patent No. CN102408483B ( NGF) human monoclonal antibody; parent antibody B: anti-NGF antibody and method of use thereof, which is the anti-human nerve growth factor (NGF) human monoclonal antibody obtained in Example 1 of Patent No. CN102746399B; parent antibody C: anti-human nerve growth The high-affinity human antibody of the factor, that is, the heavy chain and light chain CDR region amino acid sequences of the anti-human nerve growth factor (NGF) human monoclonal antibody obtained in Example 1 of Patent No. CN101827609B) are respectively integrated into the heavy chain and light chain of the human antibody. Chain scFV amino acid ...
Embodiment 2
[0044] Example 2: Construction of anti-human nerve growth factor scFv antibody expression vector and construction of recombinant engineering bacteria
[0045] 1. Construction of anti-human nerve growth factor scFv antibody expression vector
[0046] EcoRI and NotI were used to double digest the DNA sequence of the anti-human nerve growth factor scFv antibody, SEQ ID NO: 8 (synthesized by Nanjing GenScript) and the pPIC9K vector (purchased from Invitrogen, whose vector map and multiple cloning sites points such as figure 1 shown) and gel recovery, enzyme digestion results are as follows figure 2 Shown: the lane labeled scFv is cut into two bands by the anti-human nerve growth factor scFv enzyme attached to PUC57, which are 2710bp of PUC57 and 757bp of anti-human nerve growth factor scFv antibody. The lane labeled pPIC9K is 9300bp after digestion of pPIC9K; T4 ligase ligase digested vector and anti-human nerve growth factor scFv antibody DNA sequence, transformed Escherichia ...
Embodiment 3
[0059] Example 3: Induced expression and affinity chromatography purification of anti-human nerve growth factor scFv antibody
[0060] 1 induced expression
[0061] The screened positive recombinants were inoculated in 5ml YPD liquid medium, cultured overnight at 30°C, 250rpm, transferred to 100ml BMGY medium at a ratio of 1:50 the next day, cultivated at 30°C, 250rpm until A600 reached 3- 6. Collect bacteria by centrifugation and resuspend in 200ml BMMY, add 1% anhydrous methanol every day, and ferment for 4 days. The SDS-PAGE electrophoresis of the fermentation broth is as follows Figure 5 As shown, lane 1 is the fermentation broth, and lane M is the molecular weight marker. It can be seen that lane 1 has a target band at 26KD.
[0062] 2 Purification of anti-human nerve growth factor scFv antibody
[0063] (1) Centrifuge at 5000rpm for 10min at 4°C to collect the supernatant of the fermentation broth;
[0064] (2) filter the supernatant of the fermentation broth with a...
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