To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Hepatitis B Surface Antigens ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Single-Chain Antibodies ; genetics ; immunology ; metabolism

OBJECTIVES: To analyze the causes of perinatal death and related factors from the perspective of forensic medicine, and to provide references for reducing perinatal mortality and guidance for forensic identification. METHODS: A retrospective analysis was performed on 102 cases of perinatal autopsy with clinical data from the Department of Forensic Medicine of Chongqing Medical University in 2004-2016. RESULTS: Of the 102 cases of perinatal deaths, 66 （64.71%） were neonatal deaths, 24 （23.53%） were stillborn foetuses, and 12 （11.76%） were stillbirths. Among the 66 neonatal death cases, 39 （59.09%） died within 1 d, 19 （28.79%） died within 1-3 d, and 8 （12.12%） died within >3-7 d of birth. The top 3 causes of neonatal death were pulmonary diseases, congenital malformation, umbilical cord and placental abnormalities. The causes of stillborn foetus and stillbirth were mainly umbilical cord and placental abnormalities, and intrauterine asphyxia. CONCLUSIONS Pulmonary diseases, umbilical cord and placental abnormalities, and congenital malformations are the main causes of perinatal death. In order to reduce the perinatal mortality, pre-pregnancy examination and prenatal care should be strengthened, and the knowledge of pregnancy care should be popularized.
Autopsy ; Cause of Death ; Female ; Humans ; Infant ; Infant Mortality ; Pregnancy ; Retrospective Studies ; Stillbirth

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (K
Amino Acid Sequence ; Animals ; Leeches/chemistry* ; Pancreatic Elastase/antagonists & inhibitors* ; Protease Inhibitors/pharmacology* ; Proteins