Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Song, Shaozheng; He, Zhengyi; Mei, Junyan; Qi, Zhengqiang; Chen, Si; Xu, Sheng; Yuan, Yawei; Cheng, Yong

doi:10.4314/tjpr.v15i5.2

Cited by 6 publications

(24 citation statements)

References 12 publications

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“…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.…”

Section: Discussionmentioning

confidence: 91%

“…The results revealed that the exogenous genes in the genome of rabbits can be stably expressed, while also proving that homozygous transgenic animals can feasibly improve foreign gene expression levels and stability in the animal mammalian gland. The present study generated a homozygous rhPA rabbit strain and provided a reliable basis for large-scale production of thrombolytic drugs in the future (6).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Methodsmentioning

confidence: 99%

“…Next, the milk was centrifuged at 6,000 x g at 4˚C for 30 min, the supernatant was collected and the pH value was adjusted to 3.5 with 1 mol/l sodium hydroxide (50%). A total of 5 centrifugation cycles were performed at 6,000 xg at 4˚C for 10 min, the supernatant was collected after each centrifugation, and 1 mol/l sodium hydroxide was added to adjust the pH value to 4.5, 5.5, 6.5, 7.5 and 8.5 after collections of supernatant, respectively, at each successive cycle (6,33,34). Finally, the supernatant was filtered with a 0.22-µm aperture membrane filter to remove any impurities.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Jiang

Zhang

et al. 2018

Int J Mol Med

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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.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Jiang

Zhang

et al. 2018

Int J Mol Med

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Preparation of Anti-Human Podoplanin Monoclonal Antibody and its application in Immunohistochemical Diagnosis

Xie

Wang

Saeed

et al. 2018

Sci Rep

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Podoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene encoding the extracellular part of PDPN (ePDPN) was synthesized and used to expressed fusion protein ePDPN-His and GST-ePDPN, respectively, in E.coli. The purified GST-ePDPN fusion protein was mixed with QuickAntibody-Mouse5W adjuvant to immune mice, and the antiserum titer was determined by indirect ELISA. A stable cell line named 5B3 generating anti-PDPN monoclonal antibody (mAb) was obtained by hybridoma technology. The isotype of 5B3 cell line was IgG2b, and the chromosome number was 102 ± 4. The 5B3 mAb was purified successfully from ascites fluid through Protein G column, and its affinity constant was 2.94 × 108 L/mol. Besides, excellent specificity of the 5B3 mAb was further demonstrated in ELISA, western blot and immunohistochemistry experiments, suggesting that 5B3 mAb displays similar application value to D2-40, a commercial available antibody. Hence, the current study provides conclusive guidelines for preparation of other mAbs and their applications in immunohistochemistry diagnosis.

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

et al. 2019

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Poor expression is the key factor hampering the large-scale application of transgenic animal mammary gland bioreactors. A very different approach would be to evaluate the secretion of recombinant proteins into milk in response to a cleavable signal peptide of highly secreted lactoproteins. We previously reported rabbits harboring mammary gland-specific expression vector containing a fusion cDNA (goat β-lactoglobulin (BLG) signal peptide and recombinant human plasminogen activator (rhPA) coding sequences) expressed rhPA in the milk, but we did not realize the signal peptide contributed to the high rhPA concentration and did not mention it at that time. And the molecular structure and biological characteristics still remain unknown. So, rhPA in the milk was purified and characterized in the present study. rhPA was purified from the milk, and the purity of the recovered product was 98% with no loss of biological activity. Analysis of the N-terminal sequence, C-terminal sequence, and the molecular mass of purified rhPA revealed that they matched the theoretical design requirements. The active systemic anaphylaxis (ASA) reactions of the purified rhPA were negative. Taken together, these results indicated that the goat BLG signal peptide can efficiently mediate rhPA secretion into milk and was accurately cleaved off from rhPA by endogenous rabbit signal peptidase. We have reinforced the importance of a rhPA coding region fused to a cleavable heterologous signal peptide from highly secreted goat BLG to improve recombinant protein expression. It is anticipated that these findings will be widely applied to high-yield production of medically important recombinant proteins.

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Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Cited by 6 publications

References 12 publications

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

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

Preparation of Anti-Human Podoplanin Monoclonal Antibody and its application in Immunohistochemical Diagnosis

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

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