Objective:To construct the Escherichia coli (E. coli)expression system for preparation of the bone morphogenetic protein-2 (BMP2)with collagen-binding domain (CBD),and to study the methods and conditions for expression, purification and renaturation of CBD-BMP2.Methods:CBD sequence was cloned into the N-terminal of BMP2 sequence, the recombinant vector pet21b/CBD-BMP2 was constructed and transformed into E.coli BL21.The expression of recombinant protein was induced using isopropylβ-D-thiogalactopyranoside (IPTG) at 37 ℃.Ni-NTA chelate chromatography was used to purify CBD-BMP-2.Denaturing CBD-BMP2 was refolded by dilution method using ultrapure water.The refolding CBD-BMP2 was filtered through a 0.22μm microfiltration membrane for degermation.The recovery rate was calculated by the ratio of the protein concentration before and after degermation. The expression, purification, and renaturation of recombinant protein were detected by SDS-PAGE method.The concentration of CBD-BMP2 was detected by BCA assay.Results:The recombinant vector pet21b/CBD-BMP2 was successfully transformed into E.coli BL21,and the recombinant protein was expressed as inclusion bodies in E.coli.The SDS-PAGE results showed denaturing protein was dissolved in supernatant of lysis buffer with 8 mol·L-1 urea and the purified recombinant protein existed in elution buffer B with relative molecular mass about 14 000.Two bands (14 000 and 28 000)were seen in the SDS-PAGE picture,which indicated that the monomer was successfully refolded into dimer by dilution method.The concentrations of recombinant protein before and after degermation were 110 and 80 mg · L-1 , respectively, and the recovery rate was about 73%. Conclusion:The recombinant vector pet21b/CBD-BMP2 is transformed into E.coli BL21 successfully,and the recombinant CBD-BMP2 is expressed and refolded efficiently. The methods of prokaryotic expression system for preparing recombinant CBD-BMP2 protein are established.

Objective:To investigate the cause of death, survival time, and risk factors in patients diagnosed with Alzheimer′s disease (AD) at memory clinic.Methods:The patients with AD were enrolled from the memory clinic at Huashan Hospital, Fudan University between August 2002 and December 2006. Baseline data were collected and 7 rounds of telephone follow-up visits were conducted to track clinical outcomes. For death cases, the date and cause of death were recorded. Patients were divided into several subgroups based on gender and baseline cognition [Mini-Mental State Examination (MMSE) score]. Kaplan-Meier analysis and Cox proportional hazards regression models were constructed to analyze the survival time of patients and identify the risk factors.Results:A total of 499 patients were enrolled, including 199 males and 300 females. The follow-up time was 5.5(3.4,7.4) years, with longest follow-up time of 19.3 years. The median survival time after symptom onset was 11.8 years (95% CI 10.2-13.4 years), which was 12.4 years (95% CI 11.2-13.5 years) in females, significantly longer than that in males (10.2 years, 95% CI 9.6-10.8 years, logrank test, P=0.010). The median survival time after diagnosis was 8.3 years (95% CI 7.3-9.4 years), which was 8.8 years (95% CI 6.9-10.6 years) for women, significantly longer than that for men (6.8 years, 95% CI 5.7-7.9 years, logrank test, P=0.001). Patients with baseline MMSE scores≥15 ( n=265) had a median survial of 11.4 years (95% CI 9.5-13.2 years), significantly longer than those with poorer cognitive function (baseline MMSE scores<15; n=234, 7.4 years, 95% CI 6.4-8.4 years, logrank test, P<0.001). Multivariable Cox proportional hazards regression analysis showed that aging ( HR=1.027, 95% CI 1.002-1.052, P=0.034), lower body mass index (BMI; HR=1.081, 95% CI 1.023-1.139, P=0.007), lower baseline MMSE score ( HR=1.056, 95% CI 1.026-1.086, P<0.001), diabetes ( HR=1.716, 95% CI 1.076-2.735, P=0.023), and history of falls ( HR=1.536,95% CI 1.007-2.341, P=0.046) were independent risk factors for death (all P<0.05). During the follow-up, 224 of the participants died. Except for 62 cases of unknown reason, the top 6 causes of death were pneumonia (39 cases, 24.1%), cerebrovascular disease (24 cases, 14.8%), circulatory system disease(21 cases, 13.0%), multi-organ failure (17 cases, 10.5%), tumor (13 cases, 8.0%), eating disorders and malnutrition (13 cases, 8.0%). Conclusions:In the current study, the median survival time after onset for patients with AD was 11.8 years; aging, lower BMI, lower baseline cognition, comorbidities, and history of falls were independent risk factors for death; pneumonia was the most common cause of death.