key: cord-0807022-vmqvropy
authors: Rukavtsova, E. B.; Abramikhina, T. V.; Shulga, N. Ya.; Bykov, V. A.; Bur’yanov, Ya. I.
title: Tissue specific expression of hepatitis B virus surface antigen in transgenic plant cells and tissue culture
date: 2007
journal: Russ J Plant Physiol
DOI: 10.1134/s1021443707060088
sha: f517779e34858d0ead654a118f04e05e8744f23e
doc_id: 807022
cord_uid: vmqvropy

The tobacco plants (Nicotiana tabacum L.) carrying the HBsAg gene controlled by (Aocs)(3)AmasPmas, the hybrid promoter that includes regulatory elements of the agrobacterial octopine and mannopine synthase genes, as well as plants controlled by the same promoter and adh1, maize alcohol dehydrogenase gene intron were obtained. The presence of the adh1 gene intron did not significantly change the level of expression of the HBsAg gene in plants. The analysis of expression of hepatitis B virus surface antigen (HBs-antigen) in transformed plants expressing the HBsAg under the control of different promoters was made. The level of HBs-antigen in plants carrying the HBsAg gene controlled by (Aocs)(3)AmasPmas, the hybrid agrobacterium-derived promoter, was the highest in roots and made up to 0.01% of total amount of soluble protein. The level of HBs-antigen in plants carrying the HBsAg gene controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant and made up to 0.06% of the total amount of soluble protein. Hairy root and callus cultures of plants carrying the HBsAg gene and expressing the HBs-antigen were obtained.

The transgenic plant-based technologies for production of subunit vaccines of a new generation, which may become cheaper and safer alternatives to traditionally obtained vaccine preparations, are now being developed. Plant cells contain enzymatic systems of post-translational modification necessary for the assembling of monomer vaccine proteins they synthesize into the immunogenic multimer forms. Plants are fully able to synthesize target antigens that can cause an active immune response [1] of the host organism. The viral and bacterial antigens were shown to stimulate the production of immunoglobulines against the corresponding pathogens [2] [3] [4] [5] [6] [7] . Various transgenic plants are now being produced and tested in research centers worldwide as potential producers of vaccines against infection agents causing various human diseases, including the hepatitis B virus [3, 8, 9] . However, the transgenic plant-derived vaccine preparations are not yet commercially available.

Earlier we have obtained the tobacco plants expressing the synthetic gene of the hepatitis B surface antigen ( HBsAg ) controlled by single and dual 35S RNA cauliflower mosaic virus promoters (CaMV 35S and CaMV 35SS, respectively) [10, 11] . The presence of the dual 35S promoter increased expression of the antigen to the level of 0.05% of total amount of soluble protein. The transgenic potato plants expressing the HBsAg gene controlled by the same promoter and also by the patatin promoter of potato tubers were also produced. The amount of the HBs-antigen in potato tubers exceeded 1 µ g/g of tuber mass and was the highest in plants expressing the HBsAg gene controlled by the dual CaMV 35SS promoter.

To obtain plants with tissue-specific expression of the hepatitis B vaccine gene in tissue cultures and whole plants (as potential producers of the vaccine), tissue-specific promoters, especially of the hybrid agrobacterium-derived promoters (Aocs)3AmasPmas , prove to be effective. These promoters consist of the regulatory elements of the octopine synthase ( ocs ) and mannopine syntase ( mas ) genes of Agrobacterium tumefaciens. As was shown earlier, these promoters strongly induced the expression of the β -glucoronidase (GUS) gene in plants up to the level greatly exceeding that in plants controlled by the dual 35S RNA cauliflower mosaic virus promoter [12] .

The objective of this study was to obtain transgenic tobacco plants synthesizing the hepatitis B surface anti- gen controlled by ( Aocs ) 3 AmasPmas promoters and regulated by the elements of agrobacterial octopine synthase and mannopine synthase genes and also to analyze the expression profile of the HBsAg gene in different cells of the whole plant as well as that in callus and hairy root tissue cultures.

MATERIALS AND METHODS Construction of plasmids for plant transformation. pSS-HBsAg, the recombinant plasmid, carrying a synthetic gene encoding the HBsAg/mayw polypeptide [13] , was used as a gene source. The two vectors both containing the ( Aocs ) 3 AmasPmas promoter were used for cloning and plant transformation: pE1802 and pE1945 (courtesy of Dr Gelvin, Purdue University, United States) [12] . The extension of the HBsAg gene with specific sequences necessary for molecular cloning was done by means of PCR with two primers containing the Kpn I and Sal I restriction sites: 5'-CGGG-TACCATGGAAAACATTACTT and 5'-CGGTCGAC-CTATCATTAAATGTAAAC, respectively. The reaction mixture contained 0.1 µ g of pDES20 plasmid DNA as a template, 25 mM KCl, 60 mM Tris-HCl, pH 8.5, at 25 ° C, 1.5 mM MgCl 2 , 0.1% Triton X-100, 10 mM 2-mercaptoethanol, 0.2 mM dNTPs mixture (USB, United States), 0.25 µ M of each primer and 2.5 units of Taq DNA polymerase (SibEnzim, Russia).

The reaction volume was 50 µ l. The reaction started with 5 min at 94° C and was followed by 30 cycles: 1 min at 94° C, 1 min at 55° C, 1 min at 72° C, and ended with 7 min at 72° C. The Gene Amp PCR System 2400 (Perkin-Elmer, United States) was used for experiments. The amplified gene was cloned into a binary pE1802 vector for transformation of plants between the restriction sites Kpn I and Sal I. For cloning purposes, the HBsAg gene was cut out of the pSS-HBsAg plasmid between the Xho I and Cfr 9 I restriction sites and incorporated into the pE1945 vector between the same sites. The plasmid constructions obtained were used for transformation of Escherichia coli strain HB101. The constructions containing the HBsAg gene downstream the promoter were transferred into strain LBA4404 (pAL4404) of A. tumefaciens [14] by means of direct transformation [15] . The analysis of DNA of the obtained clones was done by Southern hybridization with the 32 P-labeled amplification product of the HBsAg gene [16] .

Transgenic plants. The Nicotiana tabacum L., cv. Samsun plants were cultivated in vitro in 0.5-to 1-l cultivation containers on the agar-solidified phytohormone-free MS medium [17] at 24 to 26°ë , 2 klx, and 65% relative humidity. The resulting agrobacterial strain culture was used for the infection of leaf disks according to a standard protocol [18] . The leaf disks were co-cultivated with the overnight agrobacterial cultures for two days and then transferred onto the selection MS medium containing hormones (1 mg/l of BA and 0.1 mg/l of NAA), 50 mg/l of kanamycin sulfate (Km), and 500 mg/l of cefotaxime. The regenerated shoots were passed onto the selection MS medium. Calli were obtained from leaf explants of transformed plants on the MS medium containing 3% sucrose, 0.5 mg/l BA, 2 mg/l NAA, and 50 mg/l Km. The hairy root culture was grown on the hormone-free MS medium from disk leaves of transformed plants infected with strain A4 of A. rhizogenes [19] .

Extraction of DNA from tobacco leaves. The DNA from tobacco leaves was extracted as described in [20] . Leaves were crushed in 1.5 ml Eppendorf tubes, 0.4 ml of the extraction buffer was added (containing 0.2 M Tris-HCl, pH 7.5, 0.25 M NaCl, 25 mM EDTA, and 0.5% SDS); the mixture was incubated for 1 h at room temperature and then centrifuged at 12000 rpm for clarification. An equal volume of isopropanol was added, and the DNA precipitate was dissolved in 100 µ l of the TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0). The plant DNA obtained was used as a template for PCR.

PCR analysis of the HBsAg gene. For the PCR analysis of HBsAg gene, 0.5 to 1 µ g of plant genomic DNA was used. The reaction mixture and cycle conditions were the same as above.

Immunoassay of the surface antigen level. The immunoassay of the hepatitis B virus surface antigen level in transformed plants was done as earlier described by us [12] with minor modifications. Leaves, roots, and calli of tested plants were ground in liquid nitrogen, and the extraction buffer (0.05 M sodium phosphate buffer, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 0.3% Tween 20, 0.4 mM phenylmethanesulfonyl fluoride, and 0.5% sodium ascorbate). The extract obtained was centrifuged for 20 min at 3500 rpm, the supernatant was transferred to 1.5-ml Eppendorf tubes and recentrifuged for 10 min at 12000 rpm. The "VektoHep B-HBs-antigen" test systems (JSC Vektor-Best, Russia) were used for measuring the HBs-antigen level in the supernatant. The recombinant yeast cell-derived HBsantigen [21] was used as a positive control. The assay was carried out according to the manufacturer's instructions. The total amount of soluble protein was measured according to Bradford [22] .

Mechanical leaf wounding for induction of HBsAg gene expression. Leaves were immersed into the liquid MS medium, cut with a scalpel into stripes 1 to 2 mm wide, and incubated at 24 to 26 °ë for 24 h. The injured leaves were used for immunoassay.

The recombinant plasmids used for transformation of plants are shown in Fig. 1 . These plasmids are based on the pE1802 and pE1945 vectors containing the ( Aocs ) 3 AmasPmas promoter [12] . Both plasmids are carrying HBsAg , a synthetic gene [10] . The difference between the two vectors is that the pE1945 is carrying the adh 1 the maize alcohol dehydrogenase gene intron, RUKAVTSOVA et al. but is lacking the TL enhancer. The obtained plasmid constructs carrying the HBsAg gene under the ( Aocs ) 3 AmasPmas promoter were used for the direct transformation of strain LBA4404 (pAL4404) of A. tumefaciens . Separate agrobacterial colonies were collected for extraction of plasmid DNA and its visualization in agarose gel electrophoresis. The transformed agrobacterial strains were used for the infection of tobacco leaf disks. 15 to 20 lines of transformed plants of each type were selected for subsequent molecular, genetic, and biochemical analysis.

The HBsAg gene presence in transformed plants was confirmed by means of PCR. The target DNA, which corresponds to the hepatitis B virus surface antigen, was found in all transgenic plants tested (Fig. 2) .

The immunoassay was carried out to study the expression profile of the surface antigen HBsAg in obtained plants. The HBs-antigen was found in different amounts in plants of all transgenic tobacco lines tested (see the table). The expression level of this antigen in the in vitro grown transgenic tobacco plants was up to 0.01% of total amount of soluble protein. Maximum expression of the surface HBsAg antigen was observed in roots. The genetic construct pE1945-Ag used for some of our experiments contained the alcohol dehydrogenase gene intron. The inclusion of introns into plant cells may enhance the expression of foreign genes [23] . However, in our case the presence of the adh1 gene intron did not increase the expression of the target gene. The enhancement effect seems to be dependent on the location of the intron in the genetic construct carrying the target gene.

The table shows the expression levels of the HBsantigen in plants controlled by different promoters. Thus, the expression level of the HBs-antigen in plants controlled by ( Aocs ) 3 AmasPmas, the hybrid agrobacterial promoter, reached the maximum of 0.01% of total amount of soluble protein in roots. The expression level of the HBs-antigen in plants controlled by the dual 35S RNA cauliflower mosaic virus promoter was the same in all organs of the plant, accounting for up to 0.06% of total amount of soluble protein. Therefore, (Aocs) 3 AmasPmas, the hybrid agrobacterial promoter, can be regarded as a tissue-specific element of the HBsAg gene expression, maximum expression being induced in roots. The activity of the agrobacterial mannopine synthase gene can be very different in different tissues and organs of the plant, maximum expression being observed in roots and calli [24, 25] . The expression level of the HBs-antigen in leaves of transgenic plants controlled by the (Aocs) 3 AmasPmas promoter substantially increased after wounding and reached to 0.01% of total amount of soluble protein. These results correspond with the available data on the induction of foreign gene expression in leaves of transgenic plants con-trolled by the mannopine synthase promoter under similar wounding conditions [24, 26] .

Eight lines of transgenic plants with the highest level of expression of the HBs-antigen were selected to obtain so-called hairy root tissue cultures by means of infection of leaf disks with strain A4 of A. rhizogenes culture. Hairy roots, being effectively plant tumors transformed by A. rhizogenes [19] , are a convenient system for production of secondary metabolites and recombinant proteins due to their genetic stability and fast growth in the hormone-free medium. As a result of retransformation of plants carrying the HBsAg gene with strain A4 of A. rhizogenes, hairy root cultures were obtained also carrying the HBs-antigen (Fig. 3) . The level of expression of the HBs-antigen in different lines of hairy root cultures remained the same as that in parent transgenic plants making up to 0.01% of total soluble protein.

Leaf explants of the transformed plants expressing the HBs-antigen controlled by the (Aocs) 3 AmasPmas promoter were used to obtain callus cultures. The HBsantigen expression in these cultures was also observed (Fig. 4) . This could be explained by the induction of activity of the mannopine synthase and octopine synthase promoters by a higher level of auxins in callus culture cultivated in the auxin-containing medium [24] .

It should be noted that the results of our study do not confirm the existing data on more efficient expression of foreign genes controlled by the (Aocs) 3 AmasPmas hybrid promoters that those controlled by the dual CaMV 35SS promoter [12] . In those study, the activity of the (Aocs) 3 AmasPmas promoter was judged on the basis of GUS staining assay. Another data, however, are available [27] when the researchers did not notice a considerable difference between the Bt-cry1Ia1, an 3 AmasPmas promoters. The attempts have also been made to use the (Aocs) 3 AmasPmas hybrid promoter for transformation of plants that could potentially produce the vaccine against SARS (Severe Acute Respiratory Syndrome) [28] . The expression of SARS-CoV, the target S protein, was the highest in roots of transformed tobacco and immature fruits of the transformed tomato plants.

(Aocs) 3 AmasPmas, the hybrid promoter, can be considered as a potential tool for tissue specific expression of various pharmaceutically important proteins in transformed plants and their cell cultures. (1-5) Tobacco plants transformed with pE1802-Ag (lines 8, 11, 12, 13, 21) ; (6-9) tobacco plants transformed with pE1945-Ag (lines 1, 4, 6, 9) . Average values are presented for three independent experiments; standard error in each of them did not exceed 0.0001%.

Transgenic Plants as Vaccine Production Systems

Expression of Hepatitis B Surface Antigen in Transgenic Plants

Edible Vaccines

Expression of Immunogenic Glycoprotein S Polypeptides from Transmissible Gastroenteritis Coronavirus in Transgenic Plants

Plant Expression Systems for the Production of Vaccines

Transgenic Plants as Edible Vaccines

Immunogenicity of Porcine Transmissible Gastroenteritis Virus Spike Protein Expressed in Plants

Expression of Human Hepatitis B Virus Surface Antigen Gene in Transgenic Tobacco

Polypeptides of Hepatitis B Surface Antigen Produced in Transgenic Potato

Production of Transgenic Tobacco Plants Expressing the Gene Encoding of Hepatitis B Virus Surface Antigen

Analysis of Transgenic Tobacco Plants Carring the Gene for Hepatitis B Virus Surface Antigen

Strength and Tissue Specificity of Chimeric Promoters Derived from the Octopine and Mannopine Synthase Genes

Expression of Hepatitis B Virus Surface Antigen in Transgenic Potato Plants and Its Characteristics

Transfer of Octopine T-DNA Segment to Plant Cells Mediated by Different Types of Agrobacterium Tumouror Root-Inducing Plasmids: Generality of Virulence Systems

Binary Vectors

Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Lab

Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures

Transformation of Dicotyledonous Plant Cells Using the Ni Plasmid of Agrobacterium tumefaciens and Ri Plasmid of A. rhizogenes, Plant Genetic Transformation and Gene Expression. A Laboratory Manual, Draper

Transformation of Several Species of Higher Plants by Agrobacterium rhizogenes. Sexual Transmission of the Transformed Genotype and Phenotype

A Simple and Rapid Method for the Preparation of Plant Genomic DNA for PCR Analysis

Recombinant Plasmid DNA pDES20 Codes Hepatitis B Virus Surface Antigen (HBsAg/mayw), Yeast Strain S. cerevisiae DAN-041/pDTS20 -Producer of Hepatitis B Virus Antigen and Vaccine on Its Base: RF Patent no. 2 088 664

A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding

The Effect of Intron Location on Intron-Mediated Enhancement of Gene Expression in Arabidopsis

Dual Promoter of Agrobacterium tumefaciens Mannopine Synthase Genes Is Regulated by Plant Growth Hormones

Combined Usage of Regulated Promoters and Grafting Technique in

Gene Fusions to lacZ Reveal New Expression Patterns of Chimeric Genes in Transgenic Plants

Evaluation of Bt-cry1Ia1 (cryV) Transgenic Potatoes on Two Species of Potato Tuber Moth, Phthorimaea operculella and Symmetrischema tangolias (Lepidoptera: Gelechiidae) in Peru

Severe Acute Respiratory Syndrome (SARS) S Protein Production in Plants: Development of Recombinant Vaccine

The authors thank S.V. Chernyshov for assistance in the development of the pE1945-Ag genetic construct.This study was supported by the Presidium of Russian Academy of Sciences, the program "Dynamics of Plant, Animal, and Human Genofonds" and also by Russian Foundation for Basic Research (projects nos. 05-08-01473 and 06-04-81022).