High-efficiency production of human serum albumin in the posterior silk glands of transgenic silkworms, Bombyx mori L

Abstract

Human serum albumin (HSA) is an important biological preparation with a variety of biological functions in clinical applications. In this study, the mRNA of a fusion transposase derived from the pESNT-PBase plasmid and a pBHSA plasmid containing the HSA gene under the control of a fibroin light chain (FL) promoter were co-injected into fertilized eggs. Fifty-six transgenic silkworm pedigrees expressing theexogenous recombinant HSA (rHSA) in the posterior silk glands (PSGs) with stable inheritance were successfully obtained. The SDS-PAGE and Western blot results confirmed that the rHSA was secreted into the transgenic silkworm cocoon, and the rHSA could be easily extracted with phosphate-buffered saline (PBS). In our research, the isolated highest amount rHSA constituted up to 29.1% of the total soluble protein of the cocoon shell, indicating that the transgenic silkworm produced an average of 17.4 μg/mg of rHSA in the cocoon shell. The production of soluble rHSA in the PSGs by means of generating transgenic silkworms is a novel approach, whereby a large amount of virus-free and functional HSA can be produced through the simple rearing of silkworms.

Fig 1. The structure of pBHSA and pESNT-PBase plasmids.

(A). Schematic representation of the pBHSA plasmid used in our transgenic experiment. pBL and pBR: the sequence of the left and right arms of the piggyBac transposon plasmid; FL promoter, the promoter sequence of the fibroin light chain gene; FLSP, the signal peptide sequence of the fibroin light chain gene; His6 tag, the sequential 6×His-tag; DDDDK, enterokinase recognition site; HSA CDS, the HSA coding sequence; FL polyA, the polyA signal sequence of the fibroin light chain gene; 3×P3 promoter, the artificial promoter specifically driving the marker gene expression in the eyes and nervous system; DsRed CDS, the coding sequence of the red fluorescent protein gene; SV40 polyA, the SV40 polyA signal sequence. (B). Schematic representation of the pESNT-PBase plasmid used in this transgenic experiment, which was constructed in our previous study [26].

Fig 2. The fluorescence phenotypes of the DsRed-specific expressed in the eyes of the transgene-positive silkworm.

A and C are the wild-type silkworm larvae and moths viewed under the normal light; B and D are transgene-positive silkworm larvae and moths viewed under the normal light. A' and C' are wild-type silkworm larvae and moths viewed under the red fluorescence; B' and D' are transgene-positive silkworm larvae and moths viewed under the red fluorescence. White arrows indicate the position of the eye.

Fig 3. Locations of the insertion site of the transgenic silkworm pedigrees on the chromosome.

Fig 4. Identification of rHSA gene expression in transgenic silkworm pedigrees using qRT-PCR, SDS-PAGE and Western blot, and purification of rHSA protein from the cocoon shells.

(A) The relative expression levels of the rHSA gene in the PSGs of the transgenic silkworm pedigrees on the 3^rd day of the 5^th instar were measured by qRT-PCR. WT: the wild-type silkworm Lan 10. HSA-1, HSA-2, HSA-3, HSA-4 and HSA-5: the transgene-positive silkworm pedigrees. (B) SDS-PAGE analysis of rHSA derived from the soluble protein of cocoon shells and (C) Western blot analysis of the cocoon layer. WT: the protein samples from the wild-type silkworm Lan 10. HSA-1, HSA-2, HSA-3, HSA-4 and HSA-5: the protein samples from the transgene-positive silkworm pedigrees. (D) The results of purification of the rHSA protein. S: the protein sample. W1, W2: recovered solutions after adding binding buffer. E1-E4: recovered solutions after adding elution buffer.