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Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.
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Objective:To investigate the clinical pathologic features of high-grade squamous intraepithelial lesions (HSIL) in postmenopausal women.Methods:The clinical data of hospital patients with HSIL admitted to the Department of Gynecology of The First People's Hospital of Lianyungang, Jiangsu Province from January 2019 to December 2020 were retrospectively analyzed. Patients undergoing liquid-based thin-layer cytology, high-risk human papillomavirus (HR-HPV), colposcopy, cervical biopsy and Endocervical curettage (ECC), pathological diagnosis of HSIL, followed by cold knife conization (CKC). The diagnosis and treatment process conformed to the cervical cancer diagnosis and treatment norms, and there were a total of 594 patients with no previous cervical surgery history,including 463 cases in the premenopausal group and 131 cases in the menopausal group, and the age, contact bleeding, gynecological examination, HR-HPV, liquid-based thin layer cytology, colposcopy and biopsy results, and post-cervical cone surgery pathological results were compared and analyzed, and multivariate Logistic regression analysis was carried out for statistically different factors to identify the clinical pathological characteristics of postmenopausal HSIL patients. T-test was used for the comparison between measurement data groups with normal distribution, and rank sum test was used for the comparison between measurement data groups with non normal distribution. Counting data use χ 2 test or Fisher exact probability method. Logistic regression analysis was used in multivariate analysis. Results:There was no significant difference in the positive rate of contact bleeding (12.98%(17/131)) and HR-HPV (77.86%(102/131)) between postmenopausal group and non postmenopausal group (11.45%(53/463) and 80.56% (373/463))(χ 2 values were 0.23 and 0.46; P values were 0.632 and 0.496). The proportion of cervical columnar epithelium displacement (43.51% (57/131)) and abnormal liquid-based thin-layer cytology (87.79%(115/131)) in the postmenopausal group were lower than those in the non postmenopausal group (64.36%(298/463) and 93.74%(434/463)). There was significant difference between the two groups (χ 2 values were 18.46 and 5.16; P values were < 0.001 and 0.023). The positive rate of ECC (62.60%(82/131)), cervical type Ⅲ transformation area (73.28%(96/131)), the proportion of pathological upgrading after conization (9.92%(13/131)) and the positive rate of cutting edge after conization (24.43%(32/131)) in menopausal group were higher than those in non menopausal patients (46.22%(214/463), 26.78%(124/463), 1.73%(8/463) and 5.40%(25/463)). There were significant differences between the two groups (χ 2 values were 10.95, 94.68, 20.11 and 42.62; P values were 0.001, <0.001, <0.001 and <0.001). Multivariate Logistic regression analysis showed a high proportion of cervical type Ⅲ transformational zones ( OR=6.569, 95% CI 4.130-10.446), high ECC positivity ( OR=1.978, 95% CI 1.250-3.128), the positive rate of cone incision margins was high ( OR=4.581, 95% CI 2.386-8.794), the proportion of pathological escalation after cone surgery ( OR=4.612, 95% CI 1.557-13.668) and the proportion of smooth cervical appearance were high ( OR=0.464, 95% CI 0.294-0.731), which was the clinicopathological feature of postmenopausal HSIL ( P values were <0.001, 0.004, <0.001, 0.006 and 0.001). Conclusion:There were differences in HSIL in patients before and after menopause, clinical symptoms and screening manifestations were atypical, and lesions are easy to involve the cervical canal, the positive rate of the cut margin after coneectomy was high, and the proportion of pathological escalation was high, so more aggressive intervention should be taken for women diagnosed with precancerous cervical lesions after menopause
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Objective To investigate the separate and combined detection value of serum carbohydrate antigen 125 (CA125),human epididymis protein 4 (HE-4) and D dimer (D-dimer) in the diagnosis of ovarian cancer.Methods One hundred and twenty ovarian cancer patients were selected as the observation group,one hundred and twenty patients with ovarian benign tumor as the benign group,eighty women with healthy physical examination results as the control group,then the chemiluminescence immunoassay was used to detect the expression of serum CA125 and HE-4 of the three groups,and the immune turbidimetric method was applied to examine the expression of D-dimer.At last,the diagnosis efficiency of CA125,HE-4 and D-dimer in separate and combined detection was calculated.Results CA125 in the observation group was (623.07±274.18) U/ml,HE-4 was (594.22±329.068) ng/ml and D-dimer was (418.57±276.75) ng/L,CA125 in the benign group was (45.09±32.58) U/ml,HE-4 was (97.92±57.52) ng/ml and D-dimer was (204.52±80.07) ng/L;CA125 in the control group was (40.23±28.16) U/ ml,HE-4 was (85.65±37.27) ng/ml and D-dimer was (187.57±65.74) ng/L,the differences between the three groups were statistically significant (F=122.82,89.91,54.46;P<0.05).The level of CA125 in the serum of patients with advanced stage (stage III-IV) was (586.10±278.33) U/ml,HE-4 was (437.49±238.06) ng/ml,D-dimer was (493.78±274.45) ng/L,in the early stage (stage I-II),the level of CA125 in the serum of patients with ovarian cancer was (372.12±265.31) U/ml,HE-4 was (673.64±301.68) ng/ml,D-dimer was (364.84±267.54) ng/L,and the difference was statistically significant (t=4.244,4.78,2.560,P<0.05).The detection sensitivity and specificity of CA-125 were 71.67% and 62.50% respectively.The sensitivity of HE-4 was 74.17% and the specificity was 75.00%.The sensitivity of D-dimer was 62.5% and the specificity was 60.00%.The combined detection sensitivity of the three was 80.00% and the specificity was 77.5%.Conclusion The sensitivity and specificity of the three tumor markers in combined detection were higher than those of separate detections,so the combined detection of CA125,HE-4 and D-dimer can further improve the diagnosis level of ovarian cancer.
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Objective To investigate the clinical value of 3.0T MRI for hilarcholangioc-arcinoma diagnosis.Methods T Totally48 hilarcholangiocarcinoma patients from October 2011 to June 2015 underwent diagnoses by 3.0T MRI and 64-slice spiral CT,and then the diagnosing results were compared with those by surgery and pathological examination to determine the value of 3.0T MRI for hilarcholangiocarcinoma diagnosis.Results 3.0T MRI had the positioning accuracy (100％) and qualitative accuracy (95.83％) significantly higher than the positioning accuracy (79.17％) and qualitative accuracy (81.25％) (P＜0.05).In case of hepatic duct dilatation,CT found 35 cases of hilar masses,14 cases of lymphoma,5 cases of hepatic duct wall invasion and 9 cases of portal vein invasion,while 3.0T MRI displayed 44 cases of hilar masses,26 cases of lymphoma,13 cases of hepatic duct wall invasion and 30 cases of portal vein invasion.Conclusion 3.0T MRI has high positioning and qualitative accuracies when used to diagnosing hilarcholangiocarcinoma,behaves well in displaying hepatic duct dilatation,high resolution of soft tissues,and thus is worthy promoting clinically.
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BACKGROUND:The embryonic epicardium can differentiate into myocardial cells and the cardiac stem cells with the potential of vascular smooth muscle cells, and it can differentiate into cardiac three-line cells which provide a new cellsource for the regeneration of cardiac injury. But the directed differentiation mechanisms and regulatory factors are stil unclear. OBJECTIVE:To establish the in vitro culture model of epicardial cells of mouse embryonic heart. METHODS:Embryonic hearts were dissected from the mice at pregnant 10.5-11.5 days, and the pulmonary veins, atrial tissue and left ventricle were cut off, then the embryonic hearts were transplanted into the 6-wel plates (or 35 mm dish) for culture. After cultured for 24 hours, the embryonic heart tissues were removed and cultured continuously. Phase contrast microscope was used to observe the growth characteristics of embryonic epicardial cells;the immunofluorescence technique was used to stain the specific antibody Wt-1 and Tbx18 of embryonic epicardial cells. RESULTS AND CONCLUSION:The embryonic epicardial cellmonolayers grew from tissue block edge with cobblestone-like and extended outwardly around the tissue blocks. After removed the embryonic heart, the cells grew continuously with rapid proliferation, and got fusion at 34 days. Al the embryonic epicardial cells could positively express the specific antibody Wt-1 and Tbx18 of embryonic epicardial cells. The results indicate that embryonic epicardial cells have the characteristics of rapidly growth and uniform morphology, and can express the embryonic epicardial cellspecific antibody with high purity. The successful y constructed in vitro culture models of embryonic epicardial cells provide new idea for the molecular mechanisms of directed differentiation.