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Introduction <p>Plant-based recombinant protein production provides a safe and cost-effective method for producing functional medicinal proteins. However, transgene silencing, a natural defense mechanism in plants, often reduces protein production by causing transgenes to behave differently than expected. To overcome this issue, some viruses encode a Helper-Component Proteinase (HC-Pro) that can suppress RNA silencing.</p> Objectives <p>This study evaluated the potential of a <i>Potato Virus A</i> (<i>PVA</i>)-derived HC-Pro suppressor to enhance the expression of recombinant <i>Camelus dromedarius lactoferrin</i> (<i>CdLF</i>) in <i>Nicotiana tabacum</i>.</p> Methods <p>Constructs for expressing <i>CdLF</i> were generated using the pGWB17-<i>PVA</i>-<i>HC-Pro</i> and pART27 vectors [<CitationRef CitationID="CR1">1</CitationRef>]. A cDNA library from dromedary camel mammary glands was used to amplify the <i>CdLF</i> gene using specific primers and clone it into a pTZ57R vector (Thermo Fisher Scientific, Catalog No. K1213). This was then transformed into <i>Escherichia coli</i> (DH5α). <i>N. tabacum</i> plants were transformed using <i>Agrobacterium tumefaciens</i>. PCR confirmed the presence of the CdLF gene, and expression levels were measured by qRT-PCR. A cold extraction buffer was used for protein extraction, followed by ELISA to quantify lactoferrin levels.</p> Results and conclusion <p>Co-expression of the HC-Pro suppressor effectively increased recombinant CdLF production in transgenic plants. Transcriptional increases of 5.7-fold in wild-type agroinfiltrated plants and 3.6-fold in T1 plants, compared to controls, resulted in a 3.4-fold and 1.8-fold rise in recombinant CdLF protein production, respectively. Functional analysis also confirmed that the recombinant CdLF protein had significant antimicrobial properties. Overall, this study demonstrated that the HC-Pro suppressor, derived from the <i>PVA</i> virus, significantly enhances CdLF yield and has the potential to improve plant-based recombinant protein production.</p>

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Boosting lactoferrin yields in Nicotiana tabacum via HC-Pro co-agroinfiltration

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月27日

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  • Mahbobeh Lotfi,
  • Ali Niazi,
  • Yong-Hong Liu,
  • Behzad Shahin-kaleybar,
  • Mandana Shariatzadeh,
  • Farzaneh Aram,
  • Bahman Fazeli-Nasab,
  • Javid Ahmad Parray,
  • Wen Jun Li,
  • Apurva Sharma,
  • Pankaj Kumar,
  • Rozidaini Mohd Ghazi

摘要

Introduction

Plant-based recombinant protein production provides a safe and cost-effective method for producing functional medicinal proteins. However, transgene silencing, a natural defense mechanism in plants, often reduces protein production by causing transgenes to behave differently than expected. To overcome this issue, some viruses encode a Helper-Component Proteinase (HC-Pro) that can suppress RNA silencing.

Objectives

This study evaluated the potential of a Potato Virus A (PVA)-derived HC-Pro suppressor to enhance the expression of recombinant Camelus dromedarius lactoferrin (CdLF) in Nicotiana tabacum.

Methods

Constructs for expressing CdLF were generated using the pGWB17-PVA-HC-Pro and pART27 vectors [1]. A cDNA library from dromedary camel mammary glands was used to amplify the CdLF gene using specific primers and clone it into a pTZ57R vector (Thermo Fisher Scientific, Catalog No. K1213). This was then transformed into Escherichia coli (DH5α). N. tabacum plants were transformed using Agrobacterium tumefaciens. PCR confirmed the presence of the CdLF gene, and expression levels were measured by qRT-PCR. A cold extraction buffer was used for protein extraction, followed by ELISA to quantify lactoferrin levels.

Results and conclusion

Co-expression of the HC-Pro suppressor effectively increased recombinant CdLF production in transgenic plants. Transcriptional increases of 5.7-fold in wild-type agroinfiltrated plants and 3.6-fold in T1 plants, compared to controls, resulted in a 3.4-fold and 1.8-fold rise in recombinant CdLF protein production, respectively. Functional analysis also confirmed that the recombinant CdLF protein had significant antimicrobial properties. Overall, this study demonstrated that the HC-Pro suppressor, derived from the PVA virus, significantly enhances CdLF yield and has the potential to improve plant-based recombinant protein production.