1.Academic Characteristics of Contemporary Chinese Medicine Masters in Treating Diabetic Kidney Disease Based on SrTO
Yu SUN ; Xiaodan WANG ; Yingzi CUI ; Tianying CHANG ; Fan LI ; Lisha WANG ; Chenxuan DONG ; Shoulin ZHANG ; Xing LIAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(10):258-269
ObjectiveTo explore the academic characteristics of contemporary renowned Chinese medicine masters in treating diabetic kidney disease (DKD) from the perspectives of principles, methods, formulas, and medications. MethodsIn strict accordance with the Systematic Review of Text and Opinion (SrTO) process developed by the Joanna Briggs Institute (JBI), an Australian evidence-based healthcare center, the databases including China National Knowledge Infrastructure (CNKI), VIP Database, Wanfang Data, and China Biomedical Literature Service System (SinoMed) were searched. Based on predefined inclusion and exclusion criteria, text information extraction, quality evaluation, and text information synthesis were conducted sequentially. The data were analyzed and presented in the form of text and figures. ResultsA total of 215 articles related to 43 contemporary renowned experts in the fields of Chinese medicine nephrology and endocrinology were included. The study found that the academic thoughts of these masters in the treatment of DKD are extensive, involving multiple levels such as disease understanding, therapeutic strategies, formula application, and medication use. In terms of disease understanding, the primary pathogenesis is characterized by deficiency in the root and excess in the manifestation. It is emphasized that internal factors, such as congenital endowment deficiency, interact with external factors such as improper diet, emotional disturbances, invasion of exogenous pathogens, and delayed or inappropriate treatment, to jointly induce the disease. This further gives rise to various pathogenetic theories, including obstruction of renal collaterals by blood stasis, toxin-induced damage to renal collaterals, latent wind disturbing the kidney, and internal heat leading to mass formation. In terms of therapeutic strategies and medication use, the principal treatment method is to replenish Qi and nourish Yin. Stage-based and syndrome-differentiated treatments are advocated. Flexible use of insect-derived drugs and wind-dispelling drugs is emphasized, along with proficiency in applying classical formulas and drug pairs. Integrated internal and external treatments, as well as the combined application of multiple therapeutic approaches, are commonly employed for comprehensive management. Meanwhile, the concept of "preventive treatment of disease" is upheld, and individualized long-term management of patients is advocated. ConclusionThrough the SrTO process, the academic thoughts of contemporary renowned Chinese medicine masters in the treatment of DKD have been systematically and standardly synthesized, providing a scientific and standardized basis for future theoretical exploration.
2.Academic Characteristics of Contemporary Chinese Medicine Masters in Treating Diabetic Kidney Disease Based on SrTO
Yu SUN ; Xiaodan WANG ; Yingzi CUI ; Tianying CHANG ; Fan LI ; Lisha WANG ; Chenxuan DONG ; Shoulin ZHANG ; Xing LIAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(10):258-269
ObjectiveTo explore the academic characteristics of contemporary renowned Chinese medicine masters in treating diabetic kidney disease (DKD) from the perspectives of principles, methods, formulas, and medications. MethodsIn strict accordance with the Systematic Review of Text and Opinion (SrTO) process developed by the Joanna Briggs Institute (JBI), an Australian evidence-based healthcare center, the databases including China National Knowledge Infrastructure (CNKI), VIP Database, Wanfang Data, and China Biomedical Literature Service System (SinoMed) were searched. Based on predefined inclusion and exclusion criteria, text information extraction, quality evaluation, and text information synthesis were conducted sequentially. The data were analyzed and presented in the form of text and figures. ResultsA total of 215 articles related to 43 contemporary renowned experts in the fields of Chinese medicine nephrology and endocrinology were included. The study found that the academic thoughts of these masters in the treatment of DKD are extensive, involving multiple levels such as disease understanding, therapeutic strategies, formula application, and medication use. In terms of disease understanding, the primary pathogenesis is characterized by deficiency in the root and excess in the manifestation. It is emphasized that internal factors, such as congenital endowment deficiency, interact with external factors such as improper diet, emotional disturbances, invasion of exogenous pathogens, and delayed or inappropriate treatment, to jointly induce the disease. This further gives rise to various pathogenetic theories, including obstruction of renal collaterals by blood stasis, toxin-induced damage to renal collaterals, latent wind disturbing the kidney, and internal heat leading to mass formation. In terms of therapeutic strategies and medication use, the principal treatment method is to replenish Qi and nourish Yin. Stage-based and syndrome-differentiated treatments are advocated. Flexible use of insect-derived drugs and wind-dispelling drugs is emphasized, along with proficiency in applying classical formulas and drug pairs. Integrated internal and external treatments, as well as the combined application of multiple therapeutic approaches, are commonly employed for comprehensive management. Meanwhile, the concept of "preventive treatment of disease" is upheld, and individualized long-term management of patients is advocated. ConclusionThrough the SrTO process, the academic thoughts of contemporary renowned Chinese medicine masters in the treatment of DKD have been systematically and standardly synthesized, providing a scientific and standardized basis for future theoretical exploration.
3.Effect and Mechanisms of Bushen Tongluo Prescription on Pulmonary Fibrosis via Inhibiting Macrophage Polarization Through Wnt3a/β-catenin Signaling Pathway
Yanxia LIANG ; Xuelian YU ; Wenwen WANG ; Guangsen LI ; Hongfei XING ; Maorong FAN ; Bin YANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(11):112-123
ObjectiveThis study aimed to investigate whether Bushen Tongluo prescription inhibits macrophage polarization by regulating the Wnt3a/β-catenin signaling pathway, thereby reducing epithelial-mesenchymal transition and excessive extracellular matrix deposition, in order to elucidate the anti-pulmonary fibrosis mechanisms of Bushen Tongluo prescription and provide a new theoretical basis for the clinical treatment of pulmonary fibrosis. MethodsFifty male Sprague-Dawley (SD) rats were randomly divided into a blank group, model group, pirfenidone group, and high- and low-dose Bushen Tongluo prescription groups. Except for the blank group, the pulmonary fibrosis model was established by intratracheal instillation of bleomycin. Intervention was initiated on day 28 after modeling. The high- and low-dose Bushen Tongluo prescription groups were administered Bushen Tongluo prescription at doses of 30.88, 15.44 g·kg-1, respectively, by intragastric gavage. The pirfenidone group was administered pirfenidone capsules at 110 mg·kg-1 by intragastric gavage. The blank and model groups were given an equal volume of normal saline by gavage, once daily for 90 days. After treatment, the level of transforming growth factor-β1 (TGF-β1) in bronchoalveolar lavage fluid (BALF) was detected by enzyme-linked immunosorbent assay (ELISA). Morphological changes in lung tissue and the collagen volume fraction were compared. The protein distribution and expression of E-cadherin, cytokeratin 19, α-smooth muscle actin (α-SMA), vimentin, collagen type Ⅰ (Col Ⅰ), and collagen type Ⅲ (Col Ⅲ) in lung tissue were detected by immunohistochemistry. The protein distribution and expression of CD68, arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), Wnt3a, and β-catenin in lung tissue were detected by immunofluorescence. The protein expression of Wnt3a and β-catenin in lung tissue was detected by Western blot, and the mRNA expression of Wnt3a and β-catenin was detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). ResultsCompared with the blank group, a large number of inflammatory cells infiltrated the airway walls, alveolar spaces, and interstitial tissue in the model group, with obvious fibrous tissue hyperplasia. The level of TGF-β1 in BALF was significantly increased. The protein expression of E-cadherin and cytokeratin 19 in lung tissue was decreased, whereas the protein expression of α-SMA, Vimentin, Wnt3a, β-catenin, Col Ⅰ, and Col Ⅲ was increased. The fluorescence-positive area ratios of CD68, Arg-1, iNOS, Wnt3a, and β-catenin in lung tissue were increased. The protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue were significantly increased (P<0.01). Compared with the model group, all treatment groups showed varying degrees of improvement in inflammatory cell infiltration and fibrous tissue hyperplasia in the airway walls, alveolar spaces, and interstitial tissue, decreased TGF-β1 levels in BALF, increased protein expression of E-cadherin and cytokeratin 19 in lung tissue, decreased protein expression of α-SMA, Vimentin, Col Ⅰ, and Col Ⅲ, decreased fluorescence-positive area ratios of CD68, Arg-1, iNOS, Wnt3a, and β-catenin in lung tissue, and decreased protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue (P<0.05, P<0.01). ConclusionBushen Tongluo prescription can improve bleomycin-induced pulmonary fibrosis in rats by inhibiting epithelial-mesenchymal transition and reducing excessive extracellular matrix deposition. The mechanism may be related to inhibition of the Wnt3a/β-catenin signaling pathway and the macrophage polarization mediated by this pathway.
4.Mechanism of Buyang Huanwutang in Inhibiting Ferroptosis and Enhancing Neurological Function Recovery After Spinal Cord Injury via GPX4-ACSL4 Axis
Luchun XU ; Guozheng JIANG ; Yukun MA ; Jiawei SONG ; Yushan GAO ; Guanlong WANG ; Jiaojiao FAN ; Yongdong YANG ; Xing YU ; Xiangsheng TANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(5):20-30
ObjectiveTo explore the mechanism by which Buyang Huanwutang regulates the glutathione peroxidase 4 (GPX4)-acyl-CoA synthetase long-chain family member 4 (ACSL4) axis to inhibit ferroptosis and promote neurological functional recovery after spinal cord injury (SCI). MethodsNinety rats were randomly divided into five groups: sham operation group, model group, low-dose Buyang Huanwutang group (12.5 g·kg-1), high-dose Buyang Huanwutang group (25 g·kg-1), and Buyang Huanwutang + inhibitor group (25 g·kg-1 + 5 g·kg-1 RSL3). The SCI model was established by using the allen method. Tissue was collected on the 7th and 28th days after operation. Motor function was assessed by using the Basso-Beattie-Bresnahan (BBB) scale. Hematoxylin-eosin (HE), Nissl, and Luxol fast blue (LFB) staining were performed to observe spinal cord histopathology. Transmission electron microscopy was used to examine mitochondrial ultrastructure. Immunofluorescence staining was used to detect the number of NeuN-positive cells and the fluorescence intensity of myelin basic protein (MBP), GPX4, and ACSL4. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to analyze the mRNA expression of GPX4 and ACSL4. Enzyme linked immunosorbent assay (ELISA) was performed to measure the levels of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD). Colorimetric assays were used to determine the iron content in spinal cord tissue. ResultsCompared to the sham operation group, the model group exhibited significantly reduced BBB scores (P<0.01), severe pathological damage in spinal cord tissue, and marked mitochondrial ultrastructural disruption. In addition, the model group showed a decrease in the number of NeuN-positive cells (P<0.01), reduced fluorescence intensity of MBP and GPX4 (P<0.01), lower levels of GSH and SOD (P<0.01), and downregulated mRNA expression of GPX4 (P<0.01). Moreover, compared to the sham operation group, the model group had elevated levels of ROS, MDA, and tissue iron content (P<0.01), along with increased fluorescence intensity and mRNA expression of ACSL4 (P<0.01). Compared with the model group and Buyang Huanwutang + inhibitor group, the Buyang Huanwutang group showed significantly improved BBB scores (P<0.05, P<0.01) and exhibited less severe spinal cord tissue damage, reduced edema and inflammatory cell infiltration, increased neuronal survival, and more intact myelin structures. Additionally, mitochondrial ultrastructure was significantly improved in the Buyang Huanwutang group. Compared to the model group and Buyang Huanwutang + inhibitor group, the Buyang Huanwutang group significantly increased the number of NeuN-positive cells and the fluorescence intensity of MBP (P<0.05, P<0.01). Furthermore, Buyang Huanwutang significantly increased the fluorescence intensity and mRNA expression of GPX4 (P<0.01) and decreased the fluorescence intensity and mRNA expression of ACSL4 (P<0.01) compared to the model group and Buyang Huanwutang + inhibitor group. Finally, the Buyang Huanwutang group significantly decreased ROS, MDA, and tissue iron content (P<0.01) and significantly increased GSH and SOD levels (P<0.01) compared to the model group and Buyang Huanwutang + inhibitor group. ConclusionBuyang Huanwutang inhibits ferroptosis through the GPX4/ACSL4 axis, reduces secondary neuronal and myelin injury and oxidative stress, and ultimately promotes the recovery of neurological function.
5.Effects of various prone ventilation positions in patients with acute respiratory distress syndrome: a network Meta-analysis
Qiangfang YU ; Xing WEI ; Jing LI ; Tianbo LI ; Yong WANG ; Sijia GU ; Liqin CHEN
Chinese Journal of Postgraduates of Medicine 2025;48(5):398-405
Objective:To compare the effects of 5 prone ventilation positions in patients with acute respiratory distress syndrome (ARDS) with network Meta-analysis.Methods:The randomized controlled trials on various prone ventilation positions in patients with ARDS were retrieved from Cochrane Library, EMbase, CINAHL, PubMed, Web of Science, SinoMed, CNKI, Chinese Medical Association Guideline Database, Wanfang Data, China Biology Medicine disc, VIP Information and Chinese Clinical Trial Registry from their inception to December 31, 2023. Literature screening, quality assessment and data extraction were done following the inclusion and exclusion criteria. Network Meta-analysis of outcome indicators was done using Stata 18.0.Results:A total of 19 articles, covering 1 284 patients and 5 prone ventilation positions (0°, 10°, 30°, 25° and 45°) were included. Cochrane risk assessment results showed that 15 articles were grade B and 4 articles were grade C. In direct comparisons, heterogeneity was acceptable ( I2≤50% and P≥0.1). In network analysis, the global and local consistency test result showed good consistency ( P>0.05). Network Meta-analysis result showed that the incidences of pressure injury in patients with 10°, 25°, 30° and 45° prone ventilation were significantly lower than that in patients with 0° prone ventilation, the incidence of pressure injury in patients with 25° prone ventilation was significantly lower than that in patients with 10° prone ventilation, and there were statistical differences ( P<0.05); the oxygenation index in patients with 25° prone ventilation was significantly better than that in patients with 0°, 30° and 45° prone ventilation, and there was statistical difference ( P<0.05). Conclusions:The 25° prone ventilation can both effectively improve oxygenation and maximally reduce the incidence of pressure injury in patients with ARDS.
6.Liraglutide may alleviate acetaminophen-induced liver injury by enhancing autophagy
Guo-jing XING ; Wen-bin LI ; Long-long LUO ; Li-fei WANG ; Yuan DENG ; Zhen WANG ; Zhao-jie ZHANG ; Xiao-hui YU ; Jiu-cong ZHANG
Chinese Pharmacological Bulletin 2025;41(10):1867-1875
Aim To investigate the protective effect of liraglutide(LIRA)on acetaminophen(APAP)-in-duced hepatotoxicity at the in vivo level and to reveal the underlying mechanism.Methods Forty SPF grade male C57BL/6J mice were randomly divided into the Control,LIRA(200 μg·kg-1),APAP(500 mg·kg-1),LIRA+APAP,LIRA+APAP+3-methylade-nine(3-MA,30 mg·kg-1)groups,with eight mice in each group.The mice were administered for three con-secutive days,and the materials were taken after 24 h.The general condition and body weight of mice in each group were recorded,and liver morphology was ob-served.Serum ALT and AST levels,as well as SOD ac-tivity,MDA,and GSH content in liver homogenates,were measured using biochemical assay kits.The levels of inflammatory cytokines IL-6,TNF-α,and IL-1β in serum were detected by ELISA.Liver pathological changes were assessed by HE staining,while mitochon-drial and autophagosome structures in liver tissues were observed using transmission electron microscopy.The number of PCNA-positive cells in liver tissues was e-valuated using immunohistochemical staining.The pro-tein expression levels of LC3Ⅱ,p62,Bax,Bcl-2,PC-NA,and CyclinD1 in liver tissues were determined by Western blot.Results LIRA pretreatment can im-prove the general condition of mice with acetamino-phen-induced liver injury(AILI),reduce serum ALT and AST levels,and effectively ameliorate the appear-ance and morphology of the liver as well as the patho-logical damage to liver tissue.Simultaneously,the lev-els of inflammatory cytokines IL-6,TNF-α,and IL-1βare significantly decreased;SOD activity and GSH con-tent are significantly increased,while MDA content is significantly reduced.Transmission electron microsco-py observations reveal the presence of numerous auto-phagosomes in the cytoplasm of liver tissue.Immuno-histochemical staining results indicate a significant in-crease in the number of PCNA-positive cells.Further-more,the expression of LC3Ⅱ,Bcl-2,PCNA,and Cy-clinD1 proteins in liver tissue is significantly upregulat-ed,while the expression of p62 and Bax proteins is significantly downregulated.However,after interven-tion with the autophagy inhibitor 3-MA,the aforemen-tioned protective effects of LIRA are significantly.Conclusions LIRA pretreatment can significantly im-prove liver injury in AILI mice.Its protective mecha-nism may be related to enhancing autophagy in hepato-cytes,thereby reducing oxidative stress,inflammatory response and apoptosis in liver of AILI mice.
7.Literature Study on the Evolution of TCM Syndrome Characteristics of Chronic Heart Failure From 1973 to 2023
Chidao ZHANG ; Yongxia WANG ; Mingjun ZHU ; Zuoying XING ; Boyong QIU ; Xinlu WANG ; Rui YU
World Science and Technology-Modernization of Traditional Chinese Medicine 2025;27(1):121-131
Objective To excavate the TCM syndrome of chronic heart failure from 1973 to 2023 and summarize the evolution of the characteristics of medical syndrome.Methods The relevant literature on clinical research on TCM syndromes in three databases including CNKI from January 1,1973 to October 1,2023 was searched,and SPSS 21.0 and Rstudio software were used to conduct descriptive statistical analysis,association rule analysis,and cluster analysis on the distribution characteristics of TCM syndrome types and pattern elements in the final literature.Results A total of 218 literatures,including 41604 patients,covering 29 provinces,municipalities and autonomous regions across the country,were included,of which 179 articles explicitly reported the TCM composite syndrome type,including 31123 patients.A total of 71996 pattern elements were extracted,and the common elements included qi deficiency,blood stasis,phlegm turbidity,etc.The main location of the disease include heart,liver,spleen,etc.The characteristics of TCM syndromes in different regions and different periods show certain evolution rules,from 1973 to 2023,the proportion of qi deficiency and blood stasis pattern and phlegm and stasis mutual obstruction pattern showed an upward trend.The proportion of simple false evidence increased from 1994 to 2003 compared with 1973 to 1993,but decreased from 2004 to 2023,and was lower than that from 1973 to 1993.The proportion of yang deficiency water pan syndrome showed a downward trend;The evolution of syndromes in different regions has changed from deficiency to deficiency and real mixed syndrome,for example,North China,which has the highest proportion,has changed from cardiopulmonary qi deficiency to qi deficiency and blood stasis.In recent years,the proportion of heart,liver and spleen in the chronic heart failure syndrome pattern showed an overall upward trend,the proportion of the syndrome elements of the deficiency syndrome showed a downward trend,and the proportion of the syndrome elements of the standard disease syndrome showed an upward trend.Conclusion The characteristics of chronic heart failure syndrome are always the evidence of deficiency and reality,with"deficiency and qi retention"as the core pathogenesis,and the main disease sites are the heart,liver,and spleen,and the pattern elements composed of qi deficiency,phlegm turbidity,blood stasis,and water drinking gradually occupy a dominant position,and the TCM pattern type of chronic heart failure may show a trend of"deficiency of phlegm and stasis"in the future.
8.Optimizing blood-brain barrier permeability in KRAS inhibitors:A structure-constrained molecular generation approach
Xia SHENG ; Yike GUI ; Jie YU ; Yitian WANG ; Zhenghao LI ; Xiaoya ZHANG ; Yuxin XING ; Yuqing WANG ; Zhaojun LI ; Mingyue ZHENG ; Liquan YANG ; Xutong LI
Journal of Pharmaceutical Analysis 2025;15(8):1848-1859
Kirsten rat sarcoma viral oncogene homolog(KRAS)protein inhibitors are a promising class of thera-peutics,but research on molecules that effectively penetrate the blood-brain barrier(BBB)remains limited,which is crucial for treating central nervous system(CNS)malignancies.Although molecular generation models have recently advanced drug discovery,they often overlook the complexity of bio-logical and chemical factors,leaving room for improvement.In this study,we present a structure-constrained molecular generation workflow designed to optimize lead compounds for both drug effi-cacy and drug absorption properties.Our approach utilizes a variational autoencoder(VAE)generative model integrated with reinforcement learning for multi-objective optimization.This method specifically aims to enhance BBB permeability(BBBp)while maintaining high-affinity substructures of KRAS in-hibitors.To support this,we incorporate a specialized KRAS BBB predictor based on active learning and an affinity predictor employing comparative learning models.Additionally,we introduce two novel metrics,the knowledge-integrated reproduction score(KIRS)and the composite diversity score(CDS),to assess structural performance and biological relevance.Retrospective validation with KRAS inhibitors,AMG510 and MRTX849,demonstrates the framework's effectiveness in optimizing BBBp and highlights its potential for real-world drug development applications.This study provides a robust framework for accelerating the structural enhancement of lead compounds,advancing the drug development process across diverse targets.
9.Construction of an evaluation indicator system based on Delphi method and analytic hierarchy process for accessories of patient monitor
Donglin XING ; Wei LI ; Chun PAN ; Yu WAN ; Ke XIA ; Zhenyan LU ; Yu DENG ; Hu ZHANG ; Tengfei WANG ; Gang LI
China Medical Equipment 2025;22(8):22-28
Objective:To construct an evaluation indicator system for accessories of patient monitor,so as to provide a basis for clinical management,equipment procurement,and technical improvement for accessories of medical monitor.Methods:The initial selection indicators of corresponding accessories of three types of monitoring of medical monitors,including electrocardiogram(ECG),blood oxygen saturation(SpO2)and non-invasive blood pressure(NIBP),were determined through literature research,expert consultation,and actual investigation.The Delphi method was adopted to conduct two rounds of questionnaire consultation for experts from clinical medicine,biomedical engineering and other fields in medical institutions included Sichuan Provincial People's Hospital,The Affiliated Hospital of Southwest Medical University and Medical Institute of Chengdu Institute of Metrology Verification and Testing.The evaluation indicators were screened and optimized,and the Analytic Hierarchy Process(AHP)was used to calculate the weights of each indicator.The consistency test was conducted to verify the rationality of the evaluation indicator system.Results:The evaluation indicator system for accessories of medical monitor included three first-level indicators:clinical value,cost value,and management value.The number of second-level indicators about ECG,SpO2 and NIBP of evaluation indicator system were respectively 10,9 and 8,and the number of third-level indicators of that were respectively 20,19,and 14.In the first-level indicators,the clinical value had the highest weight,with 72.49%for ECG,70.88%for SpO2 and 70.32%for NIBP.In the second-level indicators,the accuracy(28.70%for ECG,38.13%for SpO2 and 43.03%for NIBP)and safety(27.47%for ECG,26.48%for SpO2 and 23.06%for NIBP)were the core indicators.The weights of cost value and management value were between 14.62%and 17.41%,and between 12.27%and 12.89%,respectively.Conclusion:The evaluation indicator system for accessories of medical monitor integrates multi-dimensional expert opinions and quantitative analysis,highlights the priority principle for clinical performance.It can provide theoretical support for optimizing selection about accessory for medical institutions,and improving quality of monitoring,and promoting standardized management in the industry.
10.Construction of p97 mutant of Mesomycoplasma hyopneumoniae based on the homologous recombination system
Yanna WEI ; Jiying WANG ; Huan XIE ; Zhiqiang LI ; Z.A.Ishag HASSAN ; Xing XIE ; Bin XU ; Qiyan XIONG ; Zhixin FENG ; Guoqing SHAO ; Yanfei YU
Chinese Journal of Veterinary Science 2025;45(3):473-481
The aim of this study is to establish an gene editing method of Mesomycoplasma hyo-pneumoniae(Mhp)based on the homologous recombination principle.The restriction enzyme di-gestion and ligation method combined with gene synthesis were used to construct a shuttle plasmid to achieve replication in both Mhp and Escherichia coli(E.coli).The pGEM?-T vector was used as the skeleton.The oriC sequence of Mhp which can achieve the replication of the plasmid in Mhp was inserted into the vector.Sequences of the Spiroplasma promoter and puromycin resistance gene were then inserted into the above constructed plasmid to screen recombinant clones.The up-stream and downstream homologous arms of p97 were constructed to initiate homologous recombination.The recA gene of E.coli is inserted to improve the efficiency of homologous recom-bination.The obtained shuttle plasmid was then delivered into Mhp by electro-transformation or chemical transformation.A shuttle plasmid,pGEM?-Mhp-oriC-p 97,which can replicate in both Mhp and E.coli was constructed.With the transformation of this plasmid,the carried puromycin gene and recA gene can be expressed,the p97 gene can be edited.Finally,the genetically unstable p97 gene mutant was initially obtained.In this study,a tool for Mhp gene editing based on the principle of homologous recombination was established,which laid a foundation for the develop-ment of tools for studying the pathogenesis of Mhp.

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