High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro

Abstract

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

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

“…To the best of our knowledge, the present study is the first to report the expression level and stability of rhPA in the milk produced by homozygote transgenic rabbits (4). A novel PcL25/rhPA transgenic rabbit strain (K29) was established, and high expression levels of rhPA was achieved in the homozygous transgenic rabbit (5,6). Homozygous animals exhibit increased advantages, such as higher genetic stability and no need for transgenic detection of offspring rabbits.…”

“…All chemicals were purchased from Sigma-Aldrich (Merck KGaA, darmstadt, Germany) unless otherwise specified. The rhPA transgenic rabbits were generated in our lab, as previously described (5,6).…”

“…It included the effective mammary-specific expression vector PCL25/rhPA (F,E,K1 domains deletion mutant of tPA) and goat β-casein gene as regulatory elements. Subsequently, rhPA transgenic rabbits were generated through microinjection into rabbit's fertilized eggs with vector concentration at 5 ng/µl (5).…”

“…Next, the F2 transgenic rabbits were mated with normal nontransgenic rabbits to obtain F3 rabbits as a test cross. Homozygous transgenic rabbits were then screened out from the F2 generation through F2 rabbit test cross and polymerase chain reaction (PcR) according to the methods described in a previous study (5). All the rhPA-positive rabbits were identified through the PCR method.…”

“…The production of recombinant proteins through the use of bacterial hosts, mammalian cells, yeast, viruses, transgenic plants or other expression systems has numerous disadvantages, including poor biological activity, high cost and poor protein post-translation modification among other (1)(2)(3)(4). Recombinant proteins expressed in the mammary gland of transgenic animals have become a research focus as bioreactors due to their high expression, low cost, easy purification process and similar protein modification to that in the human body (4)(5)(6)(7)(8)(9)(10)(11). Therefore, the animal mammary gland is currently the most ideal expression system for the production of recombinant proteins.…”