Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus.

Tong Zhou,Yasong Zhao,Qing Li,Zongbin Cui,Bolan Zhou,Zuoyan Zhu,Guili Song,Yong Long

doi:10.3390/ijms22020687

Abstract

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

Highlights

Protein and peptide drugs are playing important roles in protection of human and animals from various diseases and their market demands are quickly increased year by year
We found that cells expressing IFN1 formed less number of plaques than the control cells transfected with pcDNA3.1 (Figure 2B), suggesting an antiviral activity of grass carp IFN1 from CIK cells
Bioreactors developed in bacteria, yeast, plants and animals always exhibit some defects in expression, activity or purification of biologically active molecules [39], so the development of novel and effective bioreactors remains a high priority task for biopharmaceutical industry

Summary

Introduction

Protein and peptide drugs are playing important roles in protection of human and animals from various diseases and their market demands are quickly increased year by year (http://www.bccresearch.com). The transgenic bioreactor can be developed by the introduction of a protein-encoding gene under the control of a promoter into the genome of microorganisms, plants or animals to effectively produce target proteins and peptide molecules with biological activities of economic values. Available bioreactors, such as bacteria [1], yeasts [2], transgenic plants [3], and transgenic animals [4], have produced a number of drugs that are widely used for medical and experimental purposes, but these bioreactors have to overcome some defects such as a lack of post-translational modification for biological activities of target proteins and peptides, high costs for the generation of bioreactors and purification of target proteins and peptides, or products that are toxic to bioreactors [5,6]. Under the control of oocyte-specific gene zp promoter, mature tilapia insulin-like growth factor was successfully expressed in zebrafish [9]

Methods

Results

Conclusion