Objective:To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods:The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results:The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values (62 kD).The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion: The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic activity of alkaline phosphatase.We anticipate that the ZZ-AP fusion protein has a potential application in immunoassay field.

Objective:To prepare the site-specific biotinylation of enhanced green fluorescence protein with double biotin molecules using Avi-tag technology.Methods:The EGFP gene was prepared by PCR and cloned into pdi-Avitag resulting the vector pEGFP-( Avitag) 2.The fusion protein EGFP-( Avitag ) 2 was expressed in E.coli DH5αand purified by employing IMAC.The site-specific biotinylation was implemented by BirA enzyme in vitro, and then was identified by competitive ELISA and Western blot.Results:The recombinant prokaryotic expression vector pEGFP-(Avitag)2 was correctly constructed,and EGFP-(Avitag)2 fusion was successfully expressed in E.coli DH5α.The results of competitive ELISA and Western blot showed that the EGFP-( Avitag) 2 could be site-specific biotinylation with double biotin molecules based on Avi-tag technology.Conclusion: The site-specific biotinylation of EGFP with double biotin molecules is successfully prepared,and we anticipate that can be used for BAS to improve the sensitivity and specificity of immunosensors.

Syndrome differentiation and treatment is an integral part of the traditional Chinese medicine （TCM） diagnostic and therapeutic system， whose development exhibits distinct stages. This paper systematically reviews the evolutionary trajectory of syndrome differentiation and treatment， from symptom-based treatment in Inner Canon of Yellow Emperor （《黄帝内经》）， to ZHANG Zhongjing's establishment of the disease-pulse-syndrome-treatment framework， through its application and development in the Ming and Qing dynasties， and finally to its recognition as a fundamental characteristic of TCM in modern times. However， the overemphasis on syndrome differentiation and treatment， coupled with a diminished focus on disease concepts， has led to its gradual alienation as the primary diagnostic and therapeutic model. The alienation mainly manifests as a tendency to prioritize syndrome over disease， resulting in the overgeneralization and limited application of the concept， and causing a lack of specificity in practice. This paper emphasizes that a correct understanding of syndrome differentiation and treatment is a necessary premise for the deve-lopment and improvement of the TCM diagnostic and therapeutic system. By integrating modern medical diagnosis to clarify disease targets and applying TCM thinking to extract common patterns of diseases， precise alignment between syndrome differentiation and treatment and modern clinical demands can be achieved， providing a reference for addressing the contemporary challenges of syndrome differentiation and treatment.