1.Preparation of immunotoxin BI7D12-PE38KDEL directed to EGFR and determination of its activity in vitro
Chunyan MAO ; Gangli AN ; Xiangling WANG ; Xiaochen ZHAI ; Huimin MENG ; Fengtao YOU ; Lin YANG
Chinese Journal of Immunology 2017;33(4):558-562,573
Objective:To prepare nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR and to examine its cytotoxicity against EGFR positive tumor cells.Methods:By using molecular cloning strategy,prokaryotic expression construct of pET28a-BI7D12-PE38KDEL was generated which consisted of nanobody 7D12 targeting EGFR in the form of a divalent fused with PE38KDEL,a truncated form of pseudomonas exotoxin A via a flexible peptide(G4S)4,and then transformed into E.coli BL21(DE3).Protein expression was induced by adding IPTG,purified by Ni-affinity column chromatography,and verified by Western blot.The binding capacity of the resulted immunotoxin to EGFR-positive cells A549,HT29,MCF-7 and EGFR-negative cells CEM,Jurkat were determined by flow cytometry assay,and its cytotoxicity against the target cells was examined.Briefly,tumor cells were treated with different dosage of the immunotoxin,and the killing efficacy of BI7D12-PE38KDEL on these cells were assessed by WST-1 assay after 72 hours.Results:The SDS-PAGE and Western blot results showed the recombinant immunotoxin BI7D12-PE38KDEL was successfully prepared,and majority of them was expressed in soluble form.BI7D12-PE38KDEL could selectively bind to EGFR-positive cells of A549,HT29,and MCF-7.More importantly,the immunotoxin exhibited much more significant killing effect on these EGFR positive cells compared to the negative control group of CEM and Jurkat cells(P<0.01).Conclusion:In the current study,the nanobody-based immunotoxin BI7D12-PE38KDEL targeting EGFR was successfully prepared and exhibited a superior inhibition effect for the growth of EGFR-positive cells.
2.Association study of ZNF804A gene polymorphism rs7597593 with schizophrenia and working memory
Xiongying CHEN ; Feng JI ; Jinguo ZHAI ; Min CHEN ; Qiumei ZHANG ; Li GAO ; Xiaochen SUN ; Zhen LIU ; Zhifang ZHANG ; Ping YU ; Jun LI
Chinese Journal of Behavioral Medicine and Brain Science 2014;23(10):893-896
Objective To assess the association between rs7597593 polymorphism of ZNF804A gene and schizophrenia,and to assess the relationship between rs7597593 polymorphism and working memory.Methods Schizophrenia patients and healthy controls were diagnosed in accordance with Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition (DSM-Ⅳ) ; 767 schizophrenia patients and 690 healthy controls were involved.Restriction fragment length polymorphism (RFLP) was carried out to genotype rs7597593 polymorphism.The cognitive function of working memory was assessed by the N-back task.Statistical analyses were carried out with SPSS19.0 software.Results The study found no significantly different genotype frequencies (x2=1.519,P=0.468) and allele frequencies(x2=1.263,P=0.261) of rs7597593 polymorphism between schizophrenia patients and healthy controls,however in the subgroup of higher IQ (IQ ≥ 110),there were significant different distributions of both genotype and allele (x2 =9.411 and 6.529; P=0.009 and 0.011 respectively).It was also found in this subgroup that risk T allele was associated with more error at 1-back task (F=6.854,P=0.009).Conclusion These results indicated that rs7597593 polymorphism was associated with individuals having spared cognitive function; carriers of T allele had worse cognitive function,which maybe a pathway that it contributes to schizophrenia.
3.Research progression on the first-line biological target therapy of advanced
FAN Shuangshuang ; ZHANG Tingting ; WANG Tian ; SHENG Binjie ; YOU Fengtao ; CHEN Dan ; ZHAI Xiaochen ; AN Gangli ; MENG Huimin ; YANG Lin
Chinese Journal of Cancer Biotherapy 2020;27(8):852-859
[Abstract] Objective: To develop a new type of CD7 chimeric antigen receptor modified T cell (CD7-CAR-T) for the treatment of CD7 positive acute myeloid leukemia (AML), and to observe its killing effect on CD7 positive AML cells. Methods: The CD7-CAR lentiviral vector was constructed based on the CD7 Nanobody sequence and costimulatory domain sequence of CD28 and 4-1BB. The lentiviral particles were packaged and used to co-transfect human T cells with protein expression blocker (PEBL), so as to prepare CD7-
CAR-T cells. Real time cellular analysis (RTCA) was used to monitor the cytotoxicity of CD7-CAR-T cells on CD7 overexpressed 293T cells. Flow cytometry assay was used to detect the effect of CD7-CAR-T cells on proliferation and cytokine secretion of AML cells with high, medium and low CD7 expressions (KG-1, HEL and Kasumi-1 cells, respectively). Results: CD7-CAR-T cell was successfully constructed and its surface expression of CD7 was successfully blocked. Compared with T cells, CD7-CAR-T cells could significantly inhibit the proliferation of CD7-293T cells and promote the release of TNF, Granzyme B and INF-γ; in addition, CD7-CAR-T cells also significantly promoted the apoptosis (t=147.1, P<0.01; t=23.57, P<0.01) and cytokine release (P<0.05 or P<0.01) in CD7 positive KG-1 and HEL cells, but had little effect on Kasumi-1 cells that only expressed minimal CD7 antigen (t=0.7058, P>0.05). Conclusion: CD7-CAR-T cells can specifically kill CD7-positive AML cells in vitro.