The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.
Cloning, Molecular ; Cysteine Endopeptidases ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Kinetics ; Viral Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo explore the characteristics of severe acute respiratory syndrome (SARS) transmission in the population base on analyzing the first imported case and the chains of transmission.

METHODSFor the first imported SARS case and cases who were transmitted by the index case, epidemiological investigations were conducted using the guidelines for surveillance and case investigation issued by the Ministry of Health. Data as the date of onset of symptoms, date of hospitalization, contact history etc. for each of the cases and their close contacts were collected and analyzed.

RESULTSThe first imported SARS case introduced to Beijing had infected 9 people within the family and at the hospital, with two of whom died of the same disease. The incubation period for that index case was 4 days, and that for the cases considered to be the secondary and tertiary generations were 7 and 8 days, respectively. The shorter the incubation period, the longer the fever would last and clinically more severe.

CONCLUSIONOne of the epidemiological characteristics of SARS in Beijing was noticed that the disease clustered in families and hospitals. Infection through droplets and close contact has been viewed as the primary mode of transmission.

Adult ; China ; epidemiology ; Contact Tracing ; Cross Infection ; transmission ; Family Health ; Female ; Humans ; Infectious Disease Transmission, Patient-to-Professional ; Male ; Middle Aged ; Severe Acute Respiratory Syndrome ; epidemiology ; transmission

Through analyzing the indication distribution of the different acupoints located at the upper limbs recorded in
Acupuncture Points ; Arm ; Goiter ; Humans ; Meridians ; Tuberculosis, Lymph Node