Production Of Functional Human Nerve Growth Factor From The Saliva Of Transgenic Pig By Using Salivary Glands As Bioreactors

Nerve growth facter(NGF)is a protein that can promote proliferation and differentiation of nerver cells.Mouse NGF(m NGF)that is purified from mouse salivary glands has been approved in China for the treatment of some nerve damage and degeneration diseases.Althoug m NGF has a high homology to hNGF,its bioactivity is significantly weaker than hNGF in human cells.Furthermore,administration of m NGF to patients may induce immunogenic responses to this exogenous protein.Therefore,highly active hNGF produced by efficient and low-cost methods could be used to replace the currently used m NGF drug.Animal salivary glands naturally express a high level of highly active NGF,indicating that they are the optimal tissues for NGF expression.Furthermore,animal salivary glands are potentially one of efficient bioreactors.This study aimed to produce hNGF from transgenic pigs by using salivary glands as bioreactors.Transgenic pig fibroblasts were selected after transfection of an hNGF expression plasmid carrying a selectable marker gene,EGFP,into pig fibroblasts.Using these transgenic fibroblasts as donor cells,4 founder transgenic pigs were produced by somatic cell nuclear transfer.All these 4 transgenic pigs expressed EGFP on their bodies and organs.PCR and Southern blot showed that hNGF gene and EGPF gene were integrated into the genome of 4 founder transgenic pigs.ELISA analysis indicated that the concentration of secreted hNGF in the saliva of transgenic pigs is between 600-1207ng/m L.Treatment of cultured PC12 cells with hNGF-containing transgenic pig's saliva demonstrated that it is functional in promoting differentiation of nerve cells.To avoid “contamination” of hNGF purified from transgenic pig's saliva by endogenously expressed pig nerve growth factor(p NGF),11 p NGF-targeting CRISPR/Cas9 expression plasmids were constructed and two plasmids with the highest p NGF-targeting efficiency were selected to co-transfect into the fibroblasts isolated from the ear tissue of hNGF transgenic pigs.Five hNGF transgenic cell colonies were selected with a null p NGF gene.These five cell colonies can be used as donor cells to produced p NGF-knockout hNGF transgenic pigs by somatic cell nuclear transfer in the future studies.In summary,the present study developed a new technique for production of human therapeutic proteins by using transgenic pig's salivary glands as bioreactors,and provided an efficient new method for synthesizing hNGF.