Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.
Animals ; Arthropod Proteins ; biosynthesis ; Baculoviridae ; Bombyx ; metabolism ; Enzyme Precursors ; biosynthesis ; Genetic Vectors ; Larva ; metabolism ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; biosynthesis

Objective: To explore the effect of exercise preconditioning (EP) on pathological cardiac hypertrophy and heart failure (HF) in pressure over-loaded experimental rats.
Methods:A total of 60 SD rats at the age of 6 weeks were randomly divided into 3 groups, n=20 in each group. Sham-operation group, Transverse aortic constriction (TAC) group and EP + TAC group. The cardiac function and structure were evaluated by echocardiography, patholgical changes and HF biomarkers were examined for EP effect at 4 and 8 weeks after TAC.
Results:Compared with Sham-operation group, the cardiac function and structure had obvious changes in the other 2 groups. Compared with TAC group, the ejection fraction in EP+ TAC group increased 15%, the heart weight index and left ventricular weight index decrease 15.7%and 20%respectively at 8 weeks after TAC, all P<0.05. Compared with Sham-operation group, the mRNA and protein expressions of ANP and BNP increased in TAC group at 4 and 8 weeks after TAC, increased in EP+TAC group at 8 week after TAC. Compared with TAC group, the mRNA expressions of ANP and BNP in EP+TAC group decreased 47%and 62%at 4 weeks after TAC, decreased 44%and 28.1%at 8 weeks after TAC, all P<0.05;the protein expression of ANP and BNP in EP+TAC group decreased 22.3%and 48%at 4 weeks after TAC, decreased 21.5%and 38.3%at 8 weeks after TAC, all P<0.01.
Conclusion: EP may improve cardiac pathological hypertrophy in pressure over-loaded rats at the early stage, and delay the heart failure process.

To develop the stable transformants of the silkworm (Bombyx mori) BmN cells that could continuously express the exogenous gene based on a non-transposon vector, an expression cassette containing human granucyto-macrophage colony-stimulating factor (hGM-CSF) gene driven by ie-1 promoter from B. mori nucleopolyhedrovirus was inserted into pIZT-V5-His to form a recombinant vector pIZT-IE-hGM-CSF, followed by transfecting the constructant into BmN cells, the stable ie-hGM-CSF cell lines were obtained after being selected with Zeocin. PCR result using the genomic DNA of the transformed BmN cells as template illustrated a specific fragment of ie-hGM-CSF, and Western blotting analysis using an antibody against hGM-CSF demonstrated a specific band with a molecular weight of 22 kDa in the transformed cells, meanwhile, the expression level of hGM-CSF determined by ELISA was about 2 814.7 pg in 10(6) transformed BmN cells.
Animals ; Animals, Genetically Modified ; Bombyx ; cytology ; genetics ; metabolism ; Cell Line ; Fibroins ; genetics ; Genetic Vectors ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic

Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.
Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; genetics ; metabolism ; Cloning, Molecular ; Fibroins ; biosynthesis ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Luminescent Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

Objective:To understand the satisfaction evaluation of contracted residents with " Internet plus" family doctor services and to explore its influencing factors.Methods:From November to December 2021, a stratified random sampling method was used to select 360 contracted residents who had utilized " Internet plus" family doctor services from community health service centers or township health clinics in various counties, cities, or districts of a city in Shandong Province for on-site questionnaire surveys. The purpose was to understand their awareness and satisfaction with the " Internet plus" family doctor services. At the same time, key informant interviews were conducted with contracted residents according to the principle of intensity sampling and information saturation, to collect their opinions and suggestions on the implementation process and model construction of " Internet plus" family doctor services. Descriptive analysis was performed on the data, and a binary logistic regression model was used to analyze the factors affecting the satisfaction of contracted residents with " Internet plus" family doctor services.Results:352 valid questionnaires were obtained. Among the respondents, 108 (30.6%) were very familiar or relatively familiar with the " Internet plus" family doctor service policy, and 251 (71.3%) expressed a high level of satisfaction with the overall " Internet plus" family doctor service. Gender, monthly income status, frequency of participation in health education activities, and satisfaction with the doctor′s daily service attitude, medical technical level, medical service charge level, and the level of communication between the service team and family members were factors affecting the satisfaction of contracted residents with " Internet plus" family doctor services ( P<0.05). Interview results showed that 38 people (95.0%) believed that there were issues with the current implementation of " Internet plus" family doctor services in China, such as the lack of an effective policy support environment, the need to improve the publicity and mobilization effects of contracting institutions, poor service cognition levels among the population, concerns about service quality, misunderstandings about internet-related concepts, network " panic" caused by electronic fraud phenomena, and cultural level differences among the elderly that limited the use of services. Conclusions:The overall satisfaction of contracted residents with the " Internet plus" family doctor services was relatively good. Various factors affected the satisfaction evaluation of contracted residents. It is important to focus on the differences in service psychological perception among contracted residents with different demographic characteristics, strengthen the construction of service teams and service platforms, and improve the public′s cognition and participation in " Internet plus" family doctor services to promote the continuous development of " Internet plus" family doctor services.