Study On The Preparation Technology Of Recombinant Human Nerve Growth Factor

Nerve growth factor(NGF)is the earliest discovered neurotrophin,which plays an extremely important role in regulating the expression of peripheral and central neuron functional characteristics.NGF is a dimer composed of two single chains of118 amino acids joined by non-covalent bonds.The monomer has a molecular weight of 13260.12 Da and contains 3 pairs of intrachain disulfide bonds.Neurotrophic keratitis(NK)is a degenerative corneal disease caused by partial or complete damage to the trigeminal nerve.This disease may even cause blindness without prompt and effective treatment.The European Medicines Agency(EMA)and the U.S.Food and Drug Administration(FDA)approved the marketing of recombinant human nerve growth factor(rh NGF)expressed from E.coli in 2017 and 2018,and its trade name is Cenegermin,which is the first clinical application of h NGF.Cenegermin has a good effect on patients with moderate to severe NK,but it is expensive as an orphan drug,which seriously affects the availability of medications for patients.This study aims to use E.coli to express rh NGF in the form of inclusion bodies,to study the preparation process of small-scale trials,and to lay the foundation for the subsequent development of generic drugs.In this paper,a comparative study of the two fusion methods for the preparation of NGF was carried out.Two kinds of genetically engineered bacterium were designed and constructed,the strain E.coli BL21(DE3)/p ET28a-(His)₆-pro-TEV-NGF(numbered as DMR486)use TEV protease to remove the N-terminal tag of the fusion protein and another strain E.coli BL21(DE3)/p ET28a-pro-R-NGF(numbered DMR635)use trypsin to remove the N-terminal tag of the fusion protein.The inclusion bodies were obtained by fermentation and induced expression of the two genetically engineered bacteria.The denaturation and renaturation of the inclusion bodies and the comparison of enzyme digestion with the corresponding tool protease showed that the process route of DMR635 was basically feasible.Secondly,the conditions for the high-density fermentation of strain DMR635 and the induced expression of the target protein were optimized in detail.By measuring the growth curve of the seed liquid,it was determined that the optimal seed liquid age was 8h.The optimal fermentation medium and optimal shaking flask fermentation conditions were initially screened Compared with the initial fermentation conditions,the optimized wet weight of recombinant bacteria increased by 1.2 times,and the wet weight of inclusion bodies increased by 1.4 times.The optimization study was carried out on the 5L fermentor,and the fermentation condition of the 5L fermentor was determined to add the final concentration of 0.20 mmol/L IPTG when the cell density OD₆₀₀ growing to 60?80,and control the dissolved oxygen at 20%?50%after induction,and the induction time was 12h?16h.Finally,the denaturation conditions and renaturation methods of inclusion bodies and the ratio of enzyme to substrate(v/v)and reaction time was explored in the digestion reaction.The optimal denaturation conditions were determined to be:4.0mol/L guanidine hydrochloride,100.0 mmol/L Tris-HCl,10.0 mmol/L EDTA,20.0mmol/L DTT,p H 9.5,and the concentration of inclusion bodies was 50.0 mg/m L.The renaturation method is pulse renaturation,and the reaction time is 5h.According to the complete disappearance of the substrate and no increase of the target product,it is determined that the optimal ratio(v/v)of enzyme to substrate is 1:200 and the reaction time is 4h.In general,this article has provided an expression method of E.coli fusion expressing rh NGF protein in the form of inclusion bodies,and explored the conditions for denaturation,renaturation and restriction enzyme digestion of inclusion bodies,and basically established a preparation process for rh NGF,which lays the foundation for the future development.