Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania major and Expression in the Chinese Hamster Ovary Cell Line

Background: TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Methods: Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blotting. Results: The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. Conclusion: The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Expression of opticin in the eyes of diabetic mice / 中华眼底病杂志

Objective To observe the opticin expression in the eyes of non-obese diabetes (NOD) mice and non-diabetic NOD mice . Methods Twenty NOD mice were divided into diabetic group (experimental group) and non-diabetic group (control group). All the mice were killed by cervical dislocation method. The eyes were harvested, and the vitreous, retina and sclera were separately collected. Western blot and real-time reverse transcription-polymerase chain reaction(RT-PCR)were respectively used to determine opticin protein and OPTC-mRNA levels. Results The opticin protein level in the vitreous and retina was lower in the experimental group(t = 4.42,4.58; P = 0. 002,0. 002), but is same in the sclera between the 2 groups (t = 0. 27, P = 0. 794). OPTC-mRNA level was vitreous> retina> selera. OPTC-mRNA levels of vitreous and retina in diabetic group were significantly Iower(t = 3.30,2. 48; P= 0. 01, 0.04); there was no statistical significant on OPTC mRNA of sclera between two groups(t = 0. 27, P = 0. 80). Conclusion Expression of opticin was suppressed in retina and vitreous of diabetic mice.