Construction of a universal recombinant expression vector that regulates the expression of human lysozyme in milk

Dan Lu,Huihua Zhang,Ning Li,Shen Liu,Shengzhe Shang,Xuezhen Yang

doi:10.15302/j-fase-2018211

Abstract

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (hLZ) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb hLZ genomic DNA, and 8.0 kb mWAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L−1, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.

Highlights

Transgenesis provides a novel platform to express foreign protein in mammary glands used as bioreactors, and Received October 31, 2017; accepted November 24, 2017The construction of a milk expression vector is an essential step for protein generation in an animal system
Our results have provided a simple approach to constructing a universal milk expression vector, and demonstrate that the resulting vector regulated the expression of human lysozyme gene (hLZ) in milk
During step 1 of the pMWAP-hLZ construction, nine clones were selected from the LB plates containing ampicillin, for PCR analyses, with two being positive

Summary

Introduction

Transgenesis provides a novel platform to express foreign protein in mammary glands used as bioreactors, and Received October 31, 2017; accepted November 24, 2017The construction of a milk expression vector is an essential step for protein generation in an animal system. Genomic DNA (gDNA) or complementary DNA (cDNA) from the foreign gene is designed to be driven by the 5′-flanking (promoter) and 3′-flanking regions of milk protein genes (such as α, β, g and k caseins, α-lactalbumin, β-lactoglobulin, and whey acidic protein)[5]. Other regulatory elements, such as signal peptide and insulator sequences, can be inserted into the vector to ensure high level and/or position-independent expression. Statistical analyses have suggested that the plasmid vector transgene often leads to low levels and unstable expression because of position effects and incomplete regulatory sequences[8]

Methods

Results

Conclusion