Efficient increase of the novel recombinant human plasminogen activator expression level and stability through the use of homozygote transgenic rabbits

Abstract

Expression efficacy of recombinant protein in current expression systems is generally low. Therefore, the expression levels of recombinant proteins in the breast milk of transgenic animals are typically low. In view of this, the present study aimed to construct homozygous transgenic rabbits with a high expression level of recombinant human plasminogen activator (rhPA) during the entire lactation period. Homozygous transgenic rabbits were obtained using an effective rhPA mammary‑specific expression vector PCL25/rhPA. The expression level and thrombolytic ability of rhPA in the milk of both homozygous and hemizygous rabbits were detected by enzyme‑linked immunosorbent and fibrin agarose plate assays. It was observed that the expression of rhPA was constant during the entire lactation period in homozygous rabbits, while the expression of rhPA declined slowly in hemizygote rhPA transgenic rabbits during the lactation period. In addition, the expression of rhPA in homozygous transgenic rabbit was ~950 µg/ml, which was markedly higher in comparison with that in hemizygote rabbits. Furthermore, increased gene copy number was observed to increase the expression level of rhPA at the same integration vector.

Accurately cleavable goat β-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland

“…Both the goat BLG signal peptide coding region and coding sequence of the K2 and P domains of t-PA were cloned into a mammary gland-specific expression vector. As expected, transgenic rabbits harboring the vector expressed rhPA in the milk at an average concentration of 1.28 mg/ml, and the concentration of rhPA in the whole lactation period of rabbits were stable (previously reported) [24,29]. Moreover, FAPA revealed that the purified rhPA had high fibrinolytic activity, and did not cause anaphylaxis by ASA which is essential for further therapeutic use.…”

“…The rhPA transgenic New Zealand White rabbit lines were generated as previously reported [24,29]. rhPA transgenic rabbits harbor a mammary gland-specific expression vector that contains an artificial cDNA.…”

“…We aimed to enhance rhPA expression through a cleavable goat β-lactoglobulin (BLG) signal peptide in the milk of transgenic rabbits, as well as to improve the molecular structure and biological characteristics for better efficacy and lower side effects as a therapeutic drug. The expression level of rhPA has been reported previously [24,29], but we did not realize that the signal peptide contributed to the high expression level and did not mention it at that time. And the molecular structure and biological characteristics remain unknown.…”

The High-Level Expression Analysis of RhPA In The RhPA/gGH Double-Transgenic Rabbits And Its Thrombolysis Activity In Vitro

“…Therefore, it is highly possible to improve the expression level of exogenous genes in transgenic animal mammary gland by using GH gene introduction. However, the current strategy to improve the expression level of tPA and rhPA genes in the mammary gland of transgenic animals is often to optimize the gene vector construction [15][16][26][27] . There are few reports on the synergistic promotion of tPA gene expression by double transgenic animals, especially the study of GH gene synergistically promoting the expression of tPA in transgenic animals at home and abroad.…”

“…They were mammalian mammary gland-specific expression vectors with goat β-casein as regulatory element and CMV as promoter (Fig. 1), which have been validated for expression recombinant protein on goat cells, mice, and individual rabbits [16,[26][27] . FSH (Ningbo Sansheng Pharmaceutical Co. Ltd.), HCG (Lizhu Pharmaceutical Co. Ltd.), Sumianxin II(Veterinary Institute of Military University), FBS(HyClone), hyaluronidase(Sigma), Zoletil50(Virbac), protease K(Sigma), M2(Sigma), M16 (Sigma), mouse anti-tPA monoclonal antibody (Santa Cruz), goat anti-mouse monoclonal antibody IgG-HRP(Santa Cruz); DNA gel purification and recovery kit was purchased from QIAGEN, various restriction enzymes and DNA polymerases were purchased from Takara Bio (Dalian) Co. Ltd., other reagents were purchased from Shanghai Pharmaceuticals, Shanghai Shenggong Bioengineering Co. Ltd., Nanjing Shengxing Biotechnology Co. Ltd.…”