Cloning And Expression Of SSA Antigen And Establishing Method For Detecting Anti-SSA

Objectives: To construct the recombinant plasmid of pPIC9k-SSA. Express the SSA protein using Pichia pastoris expression system (SMD1168), purify the expressed protein product. Investigate the characterization of SSA antigen and develop the corresponding detection methodology.Method1. Human lymphocyte leukemia (HL-60) was cultivated and separated. Then its RNA was extracted, and used as template to obtain cDNA through reverse transcription method. Finally SSA gene was amplified using cDNA as template.2. The recombinant plasmid of pPIC9k-SSA was constructed by transferring SSA gene into pPIC9k plasmid, and then transformed into the Pichia pastoris eukaryotic expression system (SMD1168) for inductive expression. The obtained product was then preliminarily purified.3. Using the obtained SSA protein as antigen, a dot immunogold filtration assay was developed to detect the SSA antibody. This is expected to provide the conventional detection project for clinics.4. The corresponding antigen characterization of the obtained SSA protein was also investigated.5. The SS patient's serum, which showed positive to SSA antibody from ENA spectrum detection, was collected as the positive reference, while the healthy people's serum, which showed negative from ENA spectrum detection, was obtained as the negative reference. They were applied in the verification of sensitivity, specificity, stability and etc. of the developed dot immunogold filtration assay. Results1. Human SSA gene was amplified by cDNA that was extracted from human lymphocyte leukemia, and the recombinant plasmid of pPIC9k-SSA was successfully constructed.2. The recombinant plasmid of pPIC9k-SSA was transformed into the Pichia pastoris eukaryotic expression system (SMD1168), and the strains having stable integration of the target gene were obtained.3. The SSA protein with relative molecular weight 50 kDa was made from inductive expression of recombinant strains, which can be integrated with the specificity of SSA antibody.4. Dot immunogold filtration assay was developed to detect the SSA antibody.ConclusionThis laboratory applied molecular cloning technology to make human SSA antigen from Pichia pastoris eukaryotic expression system (SMD1168). The preliminarily purified eukaryotic expression product (antigen) was used to develop the dot immunogold filtration assay (DIGFA) for detection of SSA antibody. The sensitivity and specificity of DIGFA were comparable to those of IB method. Due to the limit of antigen purification technology, sample number, time and etc., further investigation of the specificity of DIGFA and stability of reagent will be carried out. However, the development of DIGFA in this work is simpler, faster, and more economic compared to IB method, and is worth being promoted in the clinical application after its further improvement.