Primers bearing restriction enzyme sites for EcoR I and Hind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of the gcgasa gene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation into Escherichia coli BL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

The DagA gene and DagA(▽), which is a DagA gene encoding sequence without signal peptide, were cloned from genome DNA of Pseudoalteromonas atlantica 19262 by polymerase chain reaction (PCR). After ligation with pET21 vector, DagA and DagA(▽) were respectively expressed in Escherichia coli ER2566 using molecular chaperones DsbC and FkpA. A strain of ER2566-pET21a-DagA(▽)-DsbC was screened as a highly effective expressing system in the form of an inclusion body that had the target protein with up to 60% total bacterial protein. DagA protein was renatured and purified by dissolving it in 8 mol/l of urea, using Ni-NTA resin affinity chromatography and refolding using the urea gradient method. DagA with a molecular weight of ~30.8 kDa was identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and had the ability to digest agarose. In a pH range of 4.8–6.8, DagA maintained a bioactivity greater than 60%, with 5.8 being the optimum pH, and it exhibited activity at temperatures from 37°C to 60°C, with 55°C being the optimum temperature.

Using reverse transcription polymerase chain reaction (RT-PCR) and gene splicing by overlap extension (SOE-PCR), the DNA sequence encoding the chicken receptor activator of NF-κB ligand (RANKL) was amplified, cloned into the expressing plasmid pET-32a(+) with His-tag, and highly expressed in Escherichia coli. The purified protein was then added to primary cultures of chicken osteoclasts to observe its bioactivity. The results showed that the size of the PCR product was 1200 bp, which was consistent with the expected size, and the relative molecular weight of the induced protein was 64 kDa. Western blotting indicated that induced protein could react with anti-His antibody. It was also found that the induced protein can stimulate mature chicken osteoclasts to resorb bone.

The cDNA of bovine interleukin-18 (BoIL-18) was subcloned into pGEX6P-1 vector and transformed into Escherichia coli BL21(DE3). The recombinant protein was successfully expressed in E. coli by induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) at 0.3 mmol/l for 8 h. SDS-PAGE indicated that the BoIL-18 fusion protein, 44 kDa, was highly expressed. Densitometric scanning showed that the fusion protein expression was about 31.8% of the total bacterial protein. The biological activity of the chromatographically purified protein was assayed. The peripheral blood mononuclear cells (PBMC) proliferation test indicated that the BoIL-18 fusion protein could enhance PBMC proliferation when its concentration was more than 0.10 mg/l. Enzyme-linked immunosorbent assay (ELISA) showed that the BoIL-18 fusion protein could induce interferon (IFN)-γ production from spleen lymphocytes when it was at a concentration of more than 0.20 mg/l, and that the inducing effect of BoIL-18 fusion protein on IFN-γ was directly proportional to its concentration. This verified that the purified BoIL-18 fusion protein possessed a functional activity and could be applied in further studies.

The structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA of Actinobacillus pleuropneumoniae (APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that the apxIIA gene was expressed in Escherichia coli BL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108 cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

The β-glucosidase gene has important effects on alcoholic aroma precursors and insect resistance of the tea plant [Camellia sinensis (L.) O. Kutze]. The complete cDNA sequence of β-glucosidase of the tea plant was cloned; its full length was 1475 bp, and shared 40–60% similarity with corresponding parts of the nucleotide sequence of β-glucosidase gene from other plants. Its secondary structure contains 14.33% α-helix, 25.43% β-pleated sheet and many functional amino acid domains. The β-glucosidase gene was cloned into the pET-32a expression system and expressed at high-efficiently in Escherichia coli BL21 (DE3); the molecular weight of expressed fusion protein was 63 kDa. The results of enzymic reaction showed that the fusion protein possessed normal bioactivity, and it could catalyse the dehydration of the glycosidic bond. The soluble fusion protein was expressed mainly in the cytoplasm.