RESUMO
【Objective】 To construct the secretory expression system of insect cells to express the secretory TSHR A subunit protein in the ovarian cells of Spotoma oryzae (sf9). 【Methods】 A recombinant plasmid containing the target protein was constructed, and then the positive bacmid was screened out by the blue and white spots experiment. The verified bacmid was transfected into SF9 insect cells to obtain recombinant baculovirus. The virus was amplified, and the titer level was detected by virus plaque assay. Finally, Western blotting was used to identify the expression of the recombinant protein and optimize the expression conditions. 【Results】 During the construction of the protein expression system, PCR identification and sequencing results confirmed the correctness of the sequences of the recombinant plasmid and the recombinant bacmid. After the transfection of the bacmid, the signs of virus budding were observed in sf9 cells. The virus was collected and amplified. The titer of P1 generation virus was 2×107 pfu/m according to the plaque assay. The recombinant protein was identified by Western blotting and confirmed to be exogenous into the culture medium. The optimal condition for virus infection and protein expression was 72 h after the infection when the multiplicity of infection (MOI) was 1. 【Conclusion】 We constructed an insect cell expression system secreting TSHR 22-289 (55 ku), and the protein could be successfully glycolyzed. This system provides a preliminary basis for the construction and production of its industrial platform and also provides a useful tool for studies on TSHR protein and prevention of GO in the future.
RESUMO
Objective To evaluate the clinical significance of high frequency ultrasound in the diagnosis of duodenal obstruction in neonates.Methods Ultrasonography,clinical data and etiological diagnoses of the operation in 113 neonates with duodenal obstruction were reviewed retrospectively.The digestive tract,including stomach,duodenum,jejunoileum and colon,were examined in all patients with 8-12 MHz linear transducer before operation.Results In the 113 neonates with duodenal obstruction,63 cases were diagnosed intestinal malrotation,31 cases duodenal stenosis,14 cases annular pancreas,and 5 cases duodenal atresia.One hundred and six cases were diagnosed as duodenal obstruction by ultrasound,of which intestinal malrotation in 61 cases,duodenal stenosis in 29 cases,duodenal atresia in 4 cases,and annular pancreas in 12 cases.The diagnostic rate was 93.81% (106/113 cases),96.83% (61/63 cases),93.55% (29/31 cases),80.00% (4/5cases) and 85.71% (12/14 cases),respectively.The location of obstruction diagnosed by ultrasound was coincident with the operation in 92 cases,with a diagnostic rate of 81.42%(92/113).Conclusions High frequency ultrasound plays an important role in diagnosing the causes and location of duodenal obstruction.It can be used as the first choice of examinal methods for the neonates with duodenal obstruction.