Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Fang Zeng,Enqin Zheng,Zhenfang Wu,Guo Li,Zicong Li,Johann Urschitz,Gengyuan Cai,Chengcheng Zhao,Stefan Moisyadi,Guoling Li,Qingchun Zhu,Gelong Jiang,Huaqiang Yang,Wenchao Gao,Rui Dong,Dewu Liu

doi:10.1038/srep41270

Abstract

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

Highlights

Many species of animals secrete a large volume of saliva, often larger than the volume of milk produced
The pmPSP-human nerve growth factor (hNGF) plasmid was co-injected with the PB transposase expression helper plasmid pmPB47 into the pronuclei of 96 mouse zygotes
Following transfer of 90 microinjected embryos into the oviducts of surrogate females, 35 pups were born and 18 of them were identified as TG founder (F0) mice as the hNGF transgene and the EGFP marker gene were detected in their genomic DNA by PCR (Fig. 1B and Table 1)

Summary

Introduction

Many species of animals secrete a large volume of saliva, often larger than the volume of milk produced. We report the production of a therapeutic protein, the human nerve growth factor (hNGF), in the salivary glands of TG mice. Administration of mNGF to humans may induce immunogenic responses to this exogenous protein in patients To address these concerns, hNGF has been produced in E. coli[32,33], yeast[34], insect cells[35,36,37,38] and mammalian cells[39,40,41]. To test the feasibility of utilizing salivary glands of TG animals as efficient bioreactors for the synthesis of therapeutically important hNGF, we generated TG mice that expressed hNGF in their salivary glands, purified the secreted hNGF from their saliva and characterized the function as well as the bioactivity of purified hNGF

Methods

Results

Conclusion