The wild-type C. reinhardtii strain is CC-137. Wild-type strains were cultured in 50 mL liquid Tris–acetate–phosphate (TAP) medium at 160 rpm and 25 °C under a photoperiod of 12 h/12 h (Harris, 1989). The precipitates were collected from 1.5 mL algal fluid of the logarithmic growth stage (3,000 rpm, 5 min), then suspended and coated on solid medium, and used for biolistic bombardment transformation after being cultured for 3–4 days.

Plasmids patpX (approximately 3.95 kb) and p64D (approximately 9.8 kb) were preserved in our laboratory. patpX contains a group of polyclonal sites and carries the 5′ promoter (including 5′-UTR) of the atpA gene and the 3′ terminator of the rbcL gene, which were the C. reinhardtii chloroplast genomic endogenous genes. Plasmid p64D contains the aadA resistance gene cassette and the homologous recombinant fragment (clpP–trnL–petB–chlL–rpl23–rpl2).

In order to improve the expression efficiency of the major antigen gene-ORF2 (GenBank: AY035820.1) of PCV2, the ORF2 gene sequence was optimized and synthesized according to the preference of the chloroplast genome of C. reinhardtii by GenScript (Nanjing) Co., Ltd, Nanjing China. The NcoI and XbaI restriction sites were added at the 5′- and 3′-terminals of the codon-optimized ORF2 sequence, respectively, to facilitate the construction of a chloroplast expression vector.

Plasmid patpX and the optimized ORF2 gene were both excised by digestion with NcoI and XbaI, and the ORF2 gene was ligated to the large fragment of patpX. The ORF2 coding region was located between the chloroplast-specific promoter PatpA, 5’-UTR of atpA and the terminator of rbcL. ORF2 gene expression cassette was obtained from the chloroplast expression intermediate vector pAF25. Plasmid pAF25 was excised by digestion with EcoRV and NotI, and then the smaller fragment (approximately 1.8 kb) was blunted with the Klenow large fragment enzyme and subcloned into p64D plasmid, which was excised with EcoRV. The specific C. reinhardtii chloroplast expression vector pCR02 of the ORF2 gene was constructed.

Gold particles coated with plasmid pCR02 were bombarded into the wild-type C. reinhardtii using the biolistic device PDS100/He (Bio-Rad, California, USA) (Kindle et al., 1991). After being cultured in darkness for 12 h, the bombarded C. reinhardtii cells were washed with TAP liquid medium and distributed on TAP solid medium containing 100 μg/mL spectinomycin. Under the conditions of a photoperiod of 12 h/12 h and 25 °C for approximately 10–14 days, the cells of C. reinhardtii without resistance gradually whitened and died, and the resistant cells grew into single green colonies on the solid selective medium. The single colonies of C. reinhardtii were selected and cultured in liquid selective medium (containing 100 μg/mL spectinomycin) for PCR analysis.

The total DNA of resistant algal cells and the wild-type strain was extracted using the cetyltrimethylammonium bromide (CTAB) method. Specific primers P1 (5′-CCATGGCTATGACTTATCCAC-3′) and P2 (5′-TCTAGATTATTTTGGATTTAATGGT-3′) were designed to detect the ORF2 gene in resistant algal cells. Plasmid pCR02 was used as the positive control and wild-type C. reinhardtii as the negative control. The PCR procedures were as follows: 98 °C 3 min, 98 °C 10 s, 50 °C 10 s, 72 °C 15 s, and 72 °C 2 min, 35 cycles.

In addition, the integration site of the ORF2 gene cassette in the C. reinhardtii chloroplast genome was detected using primers P3 (5′-CCGAACAATGTTTTTATTCCTGGAG-3′) and P4 (5′-TTCGAAAGCTGTACCTAAACCTACA-3′), which are complementary to the ORF2 gene and chlL gene of the chloroplast genome of C. reinhardtii, respectively. Plasmid pCR02 was used as the positive control and wild-type C. reinhardtii as the negative control. PCR was performed as follows: 94 °C 5 min, 94 °C 30 s, 55 °C 30 s, 72 °C 2 min, and 72 °C 5 min, 35 cycles.

The total RNA of transgenic algal cells and wild-type C. reinhardtii was prepared to detect the transcription level of the ORF2 gene using a total RNA extraction kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China]. Total RNA (1 μg) was used to produce the cDNA of each line with the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time, Takara, Dalian, China), and the RT-PCR detection of cDNA was performed using ORF2-specific primers P1 and P2 under the following conditions: 94 °C 5 min, 94 °C 30 s, 55 °C 30 s, 72 °C 1 min, and 72 °C 5 min, 35 cycles.

Crude proteins of C. reinhardtii transformants and the wild-type strain were extracted as described previously (Franklin et al., 2002). Liquid algal cells (15 mL) at the logarithmic growth phase were collected through centrifugation. The precipitate was suspended in 200 μL protein lysate (750 mM Tris–HCl, pH 8.0, 15% sucrose, 100 mM β-mercaptoethanol, and 1 mM Phenylmethanesulfonyl Fluoride (PMSF)) and centrifuged for 20 min at 12,000 r/min and 4 °C. The supernatants were composed of total soluble protein (TSP) and used for Cap protein analysis. Protein concentrations were determined using the Bradford protein assay. Approximately 10 μg total soluble protein from each sample was boiled for 5 min and loaded in the wells of a 12% polyacrylamide gel.

Protein samples were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) and directly transferred onto a polyvinylidene fluoride (PVDF) membrane (300 mA, 1 h). The membrane was then saturated in 5% skim milk powder for 2 h. After washing with tris-buffered saline with tween 20 (TBST), the membrane was successively incubated with rabbit anti-PCV2 Cap polyclonal antibody (1:10,000) (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) at 4 °C overnight and then incubated with 1:5,000 diluted goat anti-rabbit IgG antibody for 1–2 h at room temperature. The target protein was visualized by adding Enhanced Chemiluminescence (ECL) chemiluminescence chromogenic solution (Yeasen Biotech Co., Ltd., Shanghai China).

The antigenicity of the Cap protein in the crude extract of C. reinhardtii transformants was determined using ELISA. The extraction of protein samples was the same as above. Crude protein samples were added to a 96-well microtiter plate that had been coated with PCV2 Cap antibody to react at 37 °C for 30 min, washed with PBST, and treated with Horseradish Peroxidase (HRP)–PCV2 Cap antibody at 37 °C for 30 min. Then, the color solution was added and incubated at 37 °C for 15 min, and the absorbance was monitored at A450 after termination of the color reaction.

The ORF2 gene sequence was optimized by GenScript (Nanjing) Co., Ltd., according to the preference of chloroplast gene expression in C. reinhardtii. A fragment of the ORF2 gene expression cassette was excised from pAF25 with EcoRV and NotI, blunted with the Klenow large fragment enzyme, and then inserted into the EcoRV site of vector p64D to construct C. reinhardtii chloroplast expression vector pCR02. The construction process is shown in Figure 1 .

Wild-type C. reinhardtii cells were bombarded with gold particles coated with plasmid pCR02 using the biolistic PDS1000/He (Bio-Rad) system (1,100 psi) and cultured on the TAP solid medium containing 100 μg/mL spectinomycin. After 10–14 days, 13 resistant green algal colonies appeared on the plates, while most algal cells were albino and died ( Figure 2 ). This indicated that the ORF2 gene and the aadA resistance gene may have been introduced into the chloroplast of C. reinhardtii, and the resistance gene has been expressed.

The total DNA of wild-type and resistant algal cells was extracted and detected using specific primers P1 and P2 of the ORF2 gene. As shown in Figure 3A , the expected ORF2 fragment, the 0.75-kb PCR product, was detected in four resistant algal cells and the positive control, but not in the wild-type algal strain. This indicated that the ORF2 gene may be integrated into the chloroplast genome of C. reinhardtii.

To verify whether the ORF2 gene was correctly integrated into the specific site of the chloroplast genome, primers P3 and P4, which are complementary to the chloroplast chlL gene and ORF2 gene, respectively, were designed to detect the insertion site. Approximately 1.4-kb products were expected in algal transformants and the positive control, but no corresponding size product appeared in the wild-type. As shown in Figure 3B , 1.4-kb fragments were obtained in algal transformants that had undergone seven rounds of spectinomycin resistance screening, but did not appear in wild-type algal cells. The results suggested that the exogenous ORF2 gene had been site-specifically integrated into the chloroplast genome of C. reinhardtii.

The transcription level of the ORF2 gene was determined through RT-PCR of the cDNA using specific primers P1 and P2 of the ORF2 gene. As indicated in Figure 4A , the single 0.5-kb band in transgenic algal transformants displayed normal transcriptional status, whereas no band was amplified in wild-type algal cells. In addition, total RNA was directly used for PCR amplification to remove the samples that were possibly contaminated with DNA. The results showed that no bands were found in the same system and conditions, thus verifying the specificity of the RT-PCR ( Figure 4B ).

To verify the expression of the Cap protein in transgenic algal transformants, total soluble protein of transformants and wild-type C. reinhardtii were extracted and analyzed using SDS–PAGE and Western blotting. Rabbit anti-PCV2 Cap polyclonal antibody and goat anti-rabbit IgG antibody were used for hybridization. As shown in Figure 5 , the 28-kDa protein was only detected in four C. reinhardtii transformants, but no protein was detected in the wild-type strain, which indicates that the Cap protein had accumulated in the chloroplast of C. reinhardtii.

The antigenicity of the Cap protein expressed in C. reinhardtii chloroplasts was detected using ELISA. Total soluble protein samples extracted from wild-type and transgenic C. reinhardtii were reacted with PCV2 Cap antibody and HRP-conjugated PCV2 Cap antibody, and the absorbance was monitored at OD ₄₅₀. The results showed that significant color reactions were observed in four C. reinhardtii transformants ( Figure 6A ) and that absorption values were all detected ( Figure 6B ), indicating that the Cap protein expressed in C. reinhardtii chloroplasts had antigenicity.