Screening of aldosterone-specific antibodies based on phage display technology / 新乡医学院学报

Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recom-binant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L-1)using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50％reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L-1 ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5#white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv(VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100％among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14％.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.Conclusion Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD1 85 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.

Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line / 生物工程学报

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Effects of different signal peptides on the secretion of human-mouse chimeric CMV-IgM / 生物工程学报

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.

Low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio predicts asymptomatic carotid plaques and their stability in high-risk stroke population / 国际脑血管病杂志

Objective To investigate the relationship between low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio (LHR) and asymptomatic carotid plaques and their stability in high-risk stroke population.Methods Between December 2012 and April 2015,a total of 39 944 permanent resident population ≥40 years were used as subjects of the survey from 11 rural communities in Haitou Town,Banzhuang Town and Tashan Town,Ganyu District,and 9 urban communities in Xinpu District and Haizhou District,Lianyungang City using epidemiological survey method of cluster sampling.Excluding those who took lipid-lowering drugs within 3 months and had a history of stroke or transient ischemic attack,6 592 people at high risk of stroke were finally screened out.Ultrasound was used to detect carotid plaques.The subjects were divided into plaque-free group and plaque group.The latter was further divided into stable plaque group and unstable plaque group.Multivariate logistic regression analysis was used to evaluate the independent risk factor for carotid plaques and their stability.The odds ratio (OR) and 95％ confidence interval (CI) were calculated.Receiver Operating Characteristic (ROC) curve was used to evaluate the prediction efficiency of LHR on carotid plaques.Results Multivariate logistic regression analysis showed that low-density lipoprotein cholesterol (LDL-C) was an independent risk factor for carotid plaques,while high-density lipoprotein cholesterol (HDL-C) was an independent protection factor of carotid plaques.Using the lowest quintile (Q1) of LHR as a reference,carotid plaque risk increased significantly with the increasing LHR (Q2:OR 1.448,95％ CI 1.082-1.937,P =0.013;Q3:OR 2.414,95％ CI 1.754-3.322,P＜0.001;Q4:OR 2.939,95％ CI 1.945-4.441,P＜0.001;Q5:OR 4.884,95％ CI 3.143-7.115,P＜0.001).ROC curve analysis showed that the area under the curve (AUC) of LHR predicting carotid plaques was 0.795 (95％ CI 0.792-0.807;P＜ 0.001),and the optimal cut-off value was 3.00 (sensitivity 68.37％,specificity 75.65％).LHR ≥3.92 (LHR in the Q4 and Q5 subgroups) was an independent risk factor for unstable carotid plaques (OR 2.915,95％ CI 2.104-4.040;P＜0.001).The AUC of the LHR predicting unstable carotid plaques was 0.658 (95％ CI 0.633-0.684;P＜0.001).Conclusions LHR was an independent predictor of carotid plaques in high-risk stroke patients.It had higher predictive value for carotid plaques,and its conversion threshold for promoting plaque formation was 3.00.When LHR was ≥3.92,there was a significant increase in the risk of unstable carotid plaques.

Clinical Observation of Acupoint Thread-embedding for Premature Ovarian Failure / 上海针灸杂志

Objective To observe the clinical efficacy of acupoint thread-embedding in treating premature ovarian failure.Method By following the visiting or admission sequence, 35 patients were randomized into a treatment group (acupoint thread-embedding group) and a control group (Western medication group). The therapeutic efficacy, and the levels of follicle stimulation hormone (FSH), luteinizing hormone (LH) and estradiol (E2) before and after the treatment were observed.Result After the treatment, the recovery rate and total effective rate were respectively 44.4% and 72.2% in the treatment group, significantly different from 0.0% and 52.9% in the control group (P0.05).Conclusion Acupoint thread-embedding can produce a satisfactory efficacy in treating premature ovarian failure.