Objective:To prepare GST-Orai2 fusion protein and to prepare polyclonal antibody against Orai2 by immunizing rabbits.To further investigate the function of Orai2,a transmembrane protein,this antibody was used to identify the Orai2 conditional gene knockout mice.Methods:ORF of Orai2 was amplified by PCR and subcloned into pGEX-6p-1 vector.After transforming BL21 (DE3) competent cells,we succeeded in inducing the expression of GST-Orai2 fusion protein using IPTG.Then the GST-Orai2 protein was purified by immobilized Glutathione affinity chromatography and identified by SDS-PAGE.A New England rabbit was immunized with the prepared fusion protein in Freund's adjuvant to prepare specific antibody.Finally,the prepared antibody was identified by Western blot by checking its titer and specificity.Furthermore,we took use of the prepared Orai2 antibody to identify Orai2 conditional gene knockout mice,with comparing to the wildtype ones in the same cage.Results:The purity of purified GST-Orai2 reached to 90% and the concentration was 0.35 mg/ml by BCA kit.We could detect Orai2 protein even in dilution of 1:10,000.Also,the prepared polyclonal antibody agianst Orai2 could detect both overexpressed and endogenous Orai2 protein in mouse-brain,without crossing reaction with Orai1.As well,we found that the Orai2 protein expression was of obvious reduction in Orai2 conditional gene knockout mice,compared with the wildtype ones in the same cage.Conclusion:We successfully obtain the purified GST-Orai2 fusion protein and prepare specific and highly sensitive polyclonal antibody against Orai2.The antibody can be used to detect overexpressed and endogenous Orai2 protein inmouse-brain specifically,and to identify Orai2 conditional gene knockout mice,without any crossing reaction with Orai1.Our work contributes a lot to the future investigation of functions of Orai2.

Immunotherapy, including immune checkpoint inhibitors, tumor vaccine therapy, oncolytic virotherapy, and adoptive cell therapy, has made remarkably breakthroughs in the field of oncology. Immune checkpoint inhibitors, which block programmed death receptor 1 or programmed death ligand 1, have been included in the first-line clinical treatment for advanced solid tumors, such as non-small cell lung cancer and malignant melanoma. However, primary or secondary drug resistance in tumors severely limits the survival benefits for patients. Immune-related adverse reactions, such as pneumonia, hypothyroidism, hypophysitis, and myocarditis, also greatly affect the quality of life of patients. Fuzheng Jiedu Huayu is an important concept guiding the prevention and treatment of tumors with traditional Chinese medicine (TCM). It is also a curative principle and therapeutic TCM method to reduce the toxicity and enhance the efficacy of immunotherapy. This article summarizes the research progress of immunotherapy and discusses how TCM reduces the toxicity and enhances the efficacy of immunotherapy, hoping to provide a reference for the integrated treatment of tumors with TCM and immunotherapy.