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  • SUN Qian-Qian,LI Fang-Zhou,ZHAO Wen-Hui
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 145-152. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1537
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    Dynamic ubiquitination in eukaryotes either enters proteins into the 26S proteasome degradation pathway or functions in signal transduction, and therefore regulates protein stability, localization and activity, thus participates in transcription, cell cycle, inflammation, tumor, immunity and other functions. Ubiquitination modification is a reversible process, which is regulated by ubiquitin ligases (E3s) and deubiquitylases (DUBs). DUBs mediate the deubiquitination of substrate proteins, regulate protein functions, and participate in various cellular processes. The protein abundance, localization and catalytic activity of deubiquitylases are strictly regulated. During the occurrence and development of tumors, many important tumor-related proteins are regulated by deubiquitylases, and dysfunction of deubiquitylases also affect DNA damage repair, apoptosis, autophagy, molecular signaling pathways and chromatin remodeling, which modulate the process of cell growth, invasion and metastasis in tumors. Therefore, DUB is an important protein family involved in tumorigenesis, and is potential drug targets. Many small molecule inhibitors have been used in the research of anti-tumor treatments. This article mainly summarizes the regulation mechanism of ubiquitin molecules, ubiquitin chain specificity, and deubiquitinating enzyme system in tumors, and provides basis for the design of clinical drug targets and diagnostic indicators.

  • Reviews
  • LI Cheng, ZHANG Hui-Wen, RONG Peng-Fei
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 153-160. https://doi.org/10.13865/j.cnki.cjbmb.2020.09.1356
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    Glypican-3 (GPC3) is an oncofetal protein anchored on the plasma membrane and highly expressed in hepatocellular carcinoma (HCC). GPC3 could be used as a biomarker for the diagnosis of HCC and the serum levels of GPC3 in liver cancer patients has a significant role for their prognosis. Moreover, GPC3 in HCC cells is immunoreactive, rendering it a suitable target for the treatment of HCC. Nowadays, several clinical trials targeting GPC3 for HCC therapy have already been conducted: new anti-GPC3 antibodies are generated; the clinical trials about its combination administration with other targeted medicines are being in progress; GPC3-targeted TRAB, GPC3 vaccines and GPC3-based chimeric antigen receptor T-cells (CAR-T) therapy are under investigation. In this review, we briefly discuss the structure of GPC3, its role in HCC pathogenesis and summarize the recent development in the clinical application of GPC3. We firmly believe that GPC3 would be a promising target for HCC therapy. And further studies focusing on GPC3 should provide us more solid evidence.
  • LI Jia, WANG Ruan-Lin, LIANG Ai-Hua
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 161-168. https://doi.org/10.13865/j.cnki.cjbmb.2020.09.1416
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    In addition to the ribosome, aminoacyl-tRNA, and mRNA, translation factors are also necessary for protein synthesis. The eukaryotic translation initiation factor 5A (eIF5A) is essential for cell viability and well conserved in all three domains during evolution. It is the only protein in eukaryotic cells that contains the unusual amino acid hypusine and the unique post-translational modification of eIF5A is strictly required for its function. eIF5A was identified in 1978 for the first time and was thought to stimulate the formation of the first peptide bond during translation initiation phase. Its involvement in the translation of polyproline-containing protein was not uncovered until 2013. With the research of over 40 years, our understanding of eIF5A function has changed dramatically. Recent ribosome profile data demonstrate that eIF5A works more generally at many ribosome stalled sites, and not limited to polyproline motif. It also enhances translation termination by facilitating peptide release. Moreover, eIF5A also indirectly regulates various cell life activities by controlling the translation of certain proteins. In this review, we provide a summary of the post-translational modification, the regulating effects during protein synthesis and autophagy as well as the relationship between eIF5A and human diseases, and explore the evolutionary conservation of eIF5A by comparing with the bacterial and archaeal orthologs, so as to provide a theoretical basis for the research in related fields.
  • ZHANG Ya, TIAN Shao-Wen
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 169-175. https://doi.org/10.13865/j.cnki.cjbmb.2020.11.1398
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    Forgetting is a critical component of the memory system. On the one hand, under physiological conditions, normal forgetting helps maintain the homeostasis of the brain memory system; on the other hand, abnormal forgetting is closely related to the occurrence and development of memory-related disorders under various neural pathological conditions. In another word, forgetting is for better memory. Forgetting of unpleasant or unnecessary memories is beneficial to update new information to adapt organisms to changing environment. Abnormal forgetting is usually associated with memory-related disorders. For example, patients with Alzheimer’s disease (AD) and epilepsy have symptoms of accelerated forgetting, and post-traumatic stress disorder (PTSD) and autistic patients cannot forget unpleasant memories. Currently, the essential relation and distinction between normal forgetting under physiological conditions and abnormal forgetting under pathological conditions are still unclear, and how to improve the memory impairment of patients by regulating the forgetting process remains to be further studied. This review mainly focuses on the involvement of Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42), neurogenesis, microglia, dopamine and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA receptors) in the regulation of forgetting under physiological conditions, and abnormal forgetting in various central pathological conditions such as AD, epilepsy, PTSD and autism. which will provide insight to the neuromolecular mechanism of forgetting and new ideas for the prevention and treatment of memory-related diseases.
  • CUI Yu-Ting,WANG Ya-Qi, WANG Pei-Chang
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 176-181. https://doi.org/10.13865/j.cnki.cjbmb.2020.11.1384
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    Alzheimer’s disease (AD), an age-associated chronic progressive neurodegeneration disorder, is characterized by progressive loss of memory, cognitive impairment and behavioral changes. The pathological hallmarks of AD are β-amyloid (Aβ) deposition, neurofibrillary tangles induced by phosphorylation of tau protein, the upregulation of inflammation and neuronal apoptosis. ?-amyloid is a polypeptide consisting of 39-42 amino acids which is produced by a series of enzymatic hydrolysis of amyloid precursor protein in neurons. Studying the regulation of the production and clearance of Aβ can effectively delay or even reverse the process of Alzheimer’s disease, which is of great importance. As the key enzyme of Aβ production, the alterations of expression level and activity of BACE1 plays an essential role in the development of AD. The aggregation of inflammatory cells around senile plaques suggests that Aβ is highly associated with neuroinflammation. Neuroinflammation-related cells participate in the clearance of Aβ and multiple cytokines regulate the level of Ab. In addition, although non-coding RNA rarely involved in the production, deposition and clearance of Aβ directly, it can regulate the production of Aβ through other pathways. In this article, we will focus on the important role of BACE1, neuroinflammation and non-coding RNA in the regulation of Aβ and review the mechanism of production and clearance of Aβ in AD.
  • CUI Yu-Qi, ZOU Chao-Xia, GAO Xu
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 182-188. https://doi.org/10.13865/j.cnki.cjbmb.2020.10.1332
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    PD-L1 (programmed cell death 1 ligand 1) is an immunosuppressive ligand which mainly expressed on tumor cells, inhibiting the activation of T lymphocytes through binding with PD-1 (programmed cell death protein 1), thus leading to immune escape. PD-1/PD-L1 immune checkpoint blockade therapy, which established based on the above mechanism, gained success in the clinical treatment of solid tumors. Various kinds of PD-L1 posttranslational modifications were identified following the in-depth studies of PD-L1, including glycosylation, phosphorylation, ubiquitination, palmitoylation, et al. Meanwhile, multiple studies indicated that posttranslational modifications governs PD-L1-mediated immune escape through regulating the protein stability and physiological functions of PD-L1, which makes posttranslational modifications of PD-L1 turns into a new “entry” point of PD-L1 studies. Drugs targeting posttranslational modifications of PD-L1 exhibited favorable applicational prospects in immunotherapy at the same time. Regulating PD-L1-mediated immune escape through intervening PD-L1 posttranslational modifications becomes a new strategy for improving the efficacy of immunotherapy. In this review, we summarized the posttranslational modifications of PD-L1 and its applicational prospects in immunotherapy, hoping to provide theoretical supports for future studies focused on PD-L1.
  • WENG Xiao-Fei, HE Ze-Zhao, LI Tao
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 189-197. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1322
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    The lineage transition between epithelium and mesenchyme is a process known as epithelial-mesenchymal transition (EMT), by which polarized epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes. EMT is commonly found in embryonic development, wound healing, organ fibrosis and tumor metastasis. EMT and mesenchymal-epithelial transition (MET) are involved in the early and late development of embryo, such as implantation, gastrulation, heart development and so on. EMT and MET are involved in the regulation of stem cell phenotype changes and cell migration, which are important mechanisms of cell differentiation and three-dimensional tissue construction. The expression of cell adhesion molecules is transformed from E-cadherin to N-cadherin, which is an important sign of EMT. By interacting with β-catenin, p120-catenin and α-catenin, E-cadherin can affect the activation of Wnt, small GTPase superfamily and other signaling pathways and regulate cytoskeleton movement. TGF, Notch, Wnt, BMP, FGF, and other signaling pathways, Snail, Twist, Zeb and other transcription factors, together with epigenetic modifying enzymes, participate in the initiation and regulation of EMT in a coordinated way. In vitro models showed that E-cadherin was involved in self-renewal of stem cells. Somatic cell reprogramming could be regarded as MET, and the expression of E-cadherin can be obtained in somatic cells after transfection of reprogramming factors. In vitro studies have found that EMT and its related molecules (such as E-cadherin, Snail, Twist, Zeb, etc.) were involved in the early triploblast differentiation and the formation of specific cell types in the late stage. The study on the mechanism of EMT will help to improve the differentiation efficiency of stem cells in vitro and promote the induction of organoids.

  • Research Papers
  • WANG Lei, SHI Ya-Wei, LU Cong-Ming
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 198-206. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1562
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    Phylloquinone is a unique cofactor of photosystem Ⅰ (PSⅠ), made up of a redoxactive naphthoquinone ring attached to a partially saturated C-20 phytyl side chain. At present, the research on the biosynthesis of phylloquinone in cyanobacteria is mainly focused on the formation of naphthoquinone ring, while there was a shortage of reports in the biosynthesis of phytyl side chain. In this study, a highly homologous protein Sll0875 was found in Synechocystis sp. PCC 6803 by homologous sequence alignment with VTE6, a kinase involved in phylloquinone biosynthesis by converting phytyl-phosphate into phytyl-diphosphate in Arabidopsis thaliana. The resulting Sll0875 mutant, called Δsll0875,accumulates none phylloquinone and tocopherol, as well as low amounts of chlorophyll content (P<0.05). The mutant had retarded growth in the absence of added glucose. Chlorophyll fluorescence, P700 absorbance changes, 77 K fluorescence emission spectra and Western blot analyses showed that in Δsll0875, PSⅠ function was impaired and accumulation of the PSⅠ complex was reduced remarkably (P<0.01), indicating that phylloquinone deficiency affected PSⅠ function, thus hindering the normal growth of cyanobacteria. Our results provide the evidence that the phytol phosphorylation pathway is essential for phylloquinone biosynthesis in cyanobacteria for the first time, and a basis for further investigate the protein synthesis, assembly and stability of PSⅠ complex in cyanobacteria.
  • YU Shu-Ting, LIU Yuan, XU Hao, LI Yang DIAO Li-Hong, ZHANG Yi, ZHU Jia-Run, HU Sheng-Wei1, LI Dong
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 207-213.
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    Extracellular matrix (ECM) is a major component of the cellular microenvironment. ECM not only provides physical support for cells, but also participates in various biological processes. In recent years, hundreds of cancer-related ECM (C-ECM) genes have been identified, and some of them can be used as biomarkers. However, rich information about C-ECM genes is scattered among thousands of publications, and there is no systematic summary of their role in tumorigenesis. Here, we present the CECMAtlas (http://biokb.ncpsb.org.cn/CECMAtlas/), a database which collects 225 C-ECM genes and the information of biological processes that they participate in through literature mining and manual curation. This database will contribute to the study of the mechanism of tumorigenesis and possible clinical application.
  • JIAO Ding-Xing, JIA Qiong, HU Shi-Xiong, YANG Wan-Yun, FAN Rui-Wen, GENG Jian-Jun
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 214-221. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1436
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    Dickkopf-3 (DKK3), as a critical inhibitor of the Wnt/β-catenin signaling pathway, may be involved in melanogenesis. In the current study, we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca. Overexpression of DKK3 in alpaca melanocytes, the expression of Wnt1, Lef1, Myc and the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TYRP2) were significantly decreased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were decreased by 80.30%, 72.17% and 64.60% (P<0.05), respectively. In contrast, in the melanocytes transfected with siRNA-DKK3 (a small interference RNA targeting DKK3), the expression of Wnt1, Lef1, Myc, MITF, TYR, TYRP1 and TYRP2 were significantly increased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were significantly increased by 1.65 folds, 1.25 folds and 1.21 folds (P<0.05), respectively. These results indicate that DKK3 regulates melanogenesis in alpaca melanocytes via the Wnt/β-catenin signaling pathway and down-regulates MITF.

  • GAO Ge, LU Yong-Quan, LIU Wei-Jin, YANG Ru-Ning, ZHANG Qi-Di, YANG Hui
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 222-228. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1514
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    Parkinson’s disease (PD) is the second major neurodegenerative disease. The pathogenesis of PD is still unclear. It is generally believed that neural damage, mitochondrial dysfunction, inflammation, oxidative stress and autophagy dysfunction caused by the transmission and aggregation of α-synuclein play an important role in the occurrence and development of PD. More and more research show that metabolic disorder is one of the pathogenesis of PD. We examined whether overexpression of α-synuclein could induce metabolic disorder in mice and the possible mechanisms. Mice were divided into two groups: Thy1-αSYN transgenic mice (TG) and the control wild-type (WT) group. The rotarod test was used to analyze motor function in mice. We detected the body weight, plasma insulin content, glucose tolerance and insulin tolerance in the two group mice. The morphology of islets in the two groups were observed by hematoxylin eosin (HE) staining, and the islets were isolated to detect the glucose-stimulated insulin secretion (GSIS). The results showed that compared with the WT group, exercise tolerance of 12-month-old TG group decreased by 23.1% (P < 0.05), body weight increased by 7% (P< 0.01), glucose tolerance decreased (P < 0.05), insulin tolerance decreased (P < 0.05), and insulin contents in the peripheral blood decreased by 20% (P < 0.05). Compared with the WT group, the levels of α-syn proteins in the pancreas of the TG group increased by 1.32 times (P < 0.05), the area of islets in the TG group decreased (P < 0.05), the number of islets decreased (P < 0.01), and the insulin secretion function decreased (P< 0.01). This study showed that the role of α-synuclein in PD is not limited to the damage of dopaminergic neurons, it also can affect metabolism and the morphology and function of peripheral organs, which provides a new theoretical basis for the pathogenesis of PD.

  • WANG Xiao-Ya, XU Qing-Bao, HE Jia-Quan
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 229-235. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1439
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    Dual-specificity phosphatase 8 (DUSP8) is a member of the dual-specificity phosphatase family, which has been reported to participate in the development of many diseases. However, it is unclear whether DUSP8 plays an important role in the inflammatory response of macrophages and related immune cells. This study aims to detect the expression of DUSP8 in lipopolysaccharide (LPS)-induced macrophage inflammatory responses and to observe the effect of DUSP8 overexpression on macrophage inflammatory responses. 100 ng/ml LPS was used to treat bone marrow-derived macrophages (BMDMs) from wild-type C57BL/6 mice at different time points. Real-time PCR and Western blotting results showed that the expression of DUSP8 was significantly reduced in BMDMs (P<0.05) and reached the peak at 12 hours. Next, DUSP8 overexpression vectors (DUSP-EGFP) and control vectors (EGFP) were transfected into BMDM. Data showed that DUSP-EGFP transfection could significantly enhance the expression of DUSP8 in BMDMs (P<0.05). Moreover, FCM data showed that the expressions of CD80 and CD86 markedly decreased in BMDMs with DUSP8 overexpression (P<0.05). Meanwhile, the neutral red uptake assay data showed that the uptake in DUSP8 overexpression group was lower than that in the control group. Furthermore, ELISA results showed DUSP8 overexpression could significantly reduce the production of IL-1β and IL-6 (P<0.05). Besides, Western blotting results showed that the phosphorylation levels of p38 MAPK and JNK decreased in BMDMs after DUSP8 overexpression (P<0.05). All together, DUSP8 overexpression could significantly ameliorate LPS-induced macrophage inflammation, which was mainly related to the reduced expression of phosphorylated p38 MAPK and JNK.

  • QIU Hui-Si, ZHOU Yi-Hai, LI Zhi-Ying, WU Hua-Zhen, FENG Zheng-Fu, WANG Xin
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 236-243. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1405
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    Long non-coding RNAs (LncRNAs), as regulators of a class of gene expression, play a key role in the development of various types of tumor. We analyzed the TCGA database and found that the expression of LncRNA AC009686.2 in breast cancer tissues was significantly higher than that in normal tissues, and was positively correlated with the poor prognosis of breast cancer patients. qRT-PCR analysis showed that the expression of LncRNA AC009686.2 in breast cancer cells was significantly up-regulated, and the expression level of LncRNA AC009686.2 in MCF7, T47D, ZR7530, BT549, HCC1937, MDA-MB-231 and SKBR3 cells was 6.58, 5.66, 7.29, 9.06, 6.89, 11.17 and 5.38 folds of that in MCF10 A cells, respectively.LncRNA AC009686.2 knockdown in MDA-MB-231 and BT549 cells which expressed relatively high LncRNA AC009686.2 significantly inhibited cell proliferation, colony formation and invasion, and induced cell G1 /S phase arrest. The clone inhibition rates of MDA-MB-231 and BT549 cells with LncRNA AC009686.2 knockdown were 0.496%, 0.438% and 0.495%, 0.353% of the control group, respectively.LncRNA AC009686.2 knockdown also down.regulated protein levels of cyclinD2 and ZEB1. However, overexpression of ZEB1 could significantly reverse the decrease of cell invasion ability caused by LncRNA AC009686.2 knockdown. We further analysed in the software JASPAR database and found that LncRNA AC009686.2 promoter had ZEB1 binding site, and overexpression of ZEB1 could down-regulate the expression level of LncRNA AC009686.2 in breast cancer cells. In conclusion, LncRNA AC009686.2 which highly expressed in breast cancer, promotes cell proliferation and invasion by up-regulating cyclinD2 and ZEB1 expression, while ZEB1 positively regulates LncRNA AC009686.2 expression. This study will provide a theoretical basis for elucidating the role of LncRNA AC009686.2 in breast cancer and related molecular mechanisms.

  • Techniques and Methods
  • JIA Qiong, WANG Rui-Wei, HU Shi-Xiong, FAN Rui-Wen
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 244-250. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1472
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    Although many microRNAs (miRNAs) are known to function as regulators of coat color and melanogenesis, the underlying molecular mechanisms of miR-100-5p governing melanogenesis were not completely known. The goal of this study was to determine the effect of miR-100-5p on melanogenesis in alpaca melanocytes. Fibroblast growth factor 21 (FGF21) is a predicted target gene of miR-100-5p and the luciferase reporter assay demonstrated that miR-100-5p regulates FGF21 by binding to its 3′ untranslated region (3′UTR). In this study, alpaca melanocytes were transfected with miR-100-5p, inhibitor and negative control plasmid. Results showed that miR-100-5p overexpression significantly decreased mRNA and protein expression of FGF21. Meanwhile, the ERK signal pathway was inhibited, with subsequent up-regulation of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2), which increased melanin production. The results suggest that miR-100-5p may regulate melanogenesis by targeting FGF21 via extracellular regulated MAP kinase (ERK) signaling pathway.
  • FAN Fen-Xia,ZHAO Wen-Xuan,KAN Biao
    Chinese Journal of Biochemistry and Molecular Biol. 2021, 37(2): 251-258. https://doi.org/10.13865/j.cnki.cjbmb.2020.12.1556
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    The bacterial two-hybrid system is a method used to detect protein-protein interaction in vivo. The method is used to analyze protein-protein interaction by supplementing the adenylate cyclase function and detecting the activity of β-galactosidase. However, some deficiencies such as complex experimental operation, low sensitivity, infeasibility for the massive samples were identified. The purpose of this study is to optimize the original bacterial two-hybrid system, and to establish an optimized system with convenient operations, batch operation, high sensitivity and real-time monitoring. In this study, we successfully constructed the plasmid pBBR-lacZ-luxCDABE with the luxCDABE reporter gene controlled by the lacZ promoter, which were transformed into this system. The results could be obtained by clone observation or testing the luminescence value rapidly and sensitively. The values of the positive control were dozens (Vibrio cholerae as hosts) to hundreds of times (Escherichia coli as hosts) of those in negative control groups, and there were significant differences between these two groups. The optimized two-hybrid system could save experimental costs, enhance our operation effectiveness and enable real-time monitoring. It was not only successfully used in Escherichia coli as hosts to analyze the interaction of proteins, but also extended the host range to Vibrio cholerae (V. cholerae). The copy number of the lacZ promoter was increased through the plasmid pBBR-lacZ-luxCDABE and the interference from non cAMP factors was weakened. As a result, the sensitivity of the modified bacterial two-hybrid system was significantly improved. These simple experimental operations also provide a favorable tool for large-scale protein-protein interaction analysis and screening of interaction proteins.