Construction of bicistronic cassette for co-expressing hepatitis B surface antigen and mouse granulocyte-macrophage colony stimulating factor as adjuvant in tobacco plant

Abstract

Context: The co-delivery of adjuvant and antigen has shown to be more effective for targeting the immune response than antigen alone. Therefore, designing an efficient bicistronic system is more assuring for production of both elements in the same tobacco cells as a plant model system. Objective: Comparing the efficient transient co-expression of hepatitis B surface antigen (HBsAg) and mouse granulocyte macrophage colony stimulating factor (mGM-CSF) in tobacco leaves by designing either mono or bicistronic cassettes. Materials and methods: Four expression cassettes containing tobacco etch virus (TEV) leader sequence were constructed with and without above genes in different orders. The cassettes were transferred into tobacco, Nicotiana tabacum L. (Solanaceae), leaves by agroinfiltration technique. The expression levels were compared using ELISA and western blotting and bioactivity of cytokine was assessed by in vitro proliferation of mouse GM-CSF-responsive progenitor cells. Results: Agroinfiltrated leaves contained recombinant HBsAg protein at 20-50 ng/mg and mGM-CSF at 0.2-4 ng/mg in both nonglycosylated and glycosylated forms. The highest expression obtained in HBsAg and mGM-CSF monocistronic co-agroinfiltrated leaves. The expression of mGM-CSF was 1.1 and 0.2 ng/mg in two different orders of bicistronic cassettes. The growth frequency of GM progenitors was approximately 1/187 cells for standard rGM-CSF and 3.2 times less activity for the plant produced. Discussion and conclusions: The recombinant mGM-CSF was produced less in bicistronic cassette than other forms; however, co-presenting of both vaccine candidate and adjuvant is confirmed and could be promising for amelioration of plant expression system as a means for vaccine production.

Keywords: GM-CSF; HBsAg; Vaccine; co-expression; plant-produced.

Figure 1.

Schematic presentation of the gene arrangements in TEV-mediated bicistronic and monocistronic constructs used for co-expression of mGM-CSF and HBsAg genes.

Figure 2.

Western blot analysis of plant-produced mouse GM-CSF. Lane 1: tobacco leaves transformed by pDE1001 vector without mGM-CSF gene as negative control; lane 2: leaves were agroinfiltrated with the vector contained mGM-CSF gene; lane 3: E. coli derived GM-CSF.

Figure 3.

Identification of positive wells based on the ability of committed progenitor cells to form colonies of mature cells. A: empty vector plant extract as negative control; B: the bone marrow microcultures containing the plant produced mGM-CSF; C: the microplate containing the plant produced mGM-CSF; D: standard GM-CSF as positive control.

Figure 4.

ELISA on plant extract to assess the expression level of GM-CSF in different bicistronic and monocistronic constructs. TEV indicates the leaves transformed by pDE1001 as negative control; TEV-GM, GM-TEV-HBs and HBs-TEV-GM are the leaves transformed by the monocistronic and bicistronic constructs, respectively (TEV-GM, TEV-HBs) indicates the leaves co-agroinfiltrated with both the GM-CSF and HBsAg monocistronic constructs.