Objective:To establish a quantitative analysis of multi-components by single marker(QAMS)for the determination of 6 alkaloid components,which is benzoylmesaconine,benzoyl-hypaconine,benzoylaconine,mesaconitine,hypaconitine,and aconitine in Zhachong Shisanwei Pills,and prove the scientificity and feasibility of the method in the quality analysis.Methods:The chromatographic separation was performed on an Agilent Eclipse Plus C18(250 mm×4.6 mm,5 μm)with gradient elution using 0.1 mol·L-1 ammonium acetate(0.5 mL of gla-cial acetic acid per 1 000 mL)(A)-acetonitrile:tetrahydrofuran(25∶15)(B),as the mobile phase(0-50 min,18%B-28%B),the detection wavelength was switched from 235 nm,the column temperature was kept at 40℃and the flow rate was 1.0 mL·min-1.The relative correction factors(fs/i)were established with the other 5 compo-nents to be measured using benzoylaconine as the internal reference,which were used to calculate the mass fraction of each component.At the same time,the mass fractions of the 6 effective constituents in Zhachong Shisanwei Pills were calculated by the external standard method(ESM).By comparing the content results of ESMand QAMS,the accura-cy of QAMS method were evaluated.Results:The relative correction factors(fs/i)of benzoylmesaconine,benzoylhyp-aconine,mesaconitine,hypaconitine,and aconitine in Mongolian medicine Zhachong Shisanwei Pills were reproduci-ble with good reproducibility,which were 0.680 4,0.450 6,0.850 8,0.676 1 and 0.757 0,the result obtained by QAMS approximated those obtained by external standard method(ESM).Conclusion:The method is simple,stable and reproducible,and can be used for the quality control of 6 alkaloid components in Zhachong Shisanwei Pills.

Objective:To establish a quantitative analysis of multi-components by single marker(QAMS)for the determination of 6 alkaloid components,which is benzoylmesaconine,benzoyl-hypaconine,benzoylaconine,mesaconitine,hypaconitine,and aconitine in Zhachong Shisanwei Pills,and prove the scientificity and feasibility of the method in the quality analysis.Methods:The chromatographic separation was performed on an Agilent Eclipse Plus C18(250 mm×4.6 mm,5 μm)with gradient elution using 0.1 mol·L-1 ammonium acetate(0.5 mL of gla-cial acetic acid per 1 000 mL)(A)-acetonitrile:tetrahydrofuran(25∶15)(B),as the mobile phase(0-50 min,18%B-28%B),the detection wavelength was switched from 235 nm,the column temperature was kept at 40℃and the flow rate was 1.0 mL·min-1.The relative correction factors(fs/i)were established with the other 5 compo-nents to be measured using benzoylaconine as the internal reference,which were used to calculate the mass fraction of each component.At the same time,the mass fractions of the 6 effective constituents in Zhachong Shisanwei Pills were calculated by the external standard method(ESM).By comparing the content results of ESMand QAMS,the accura-cy of QAMS method were evaluated.Results:The relative correction factors(fs/i)of benzoylmesaconine,benzoylhyp-aconine,mesaconitine,hypaconitine,and aconitine in Mongolian medicine Zhachong Shisanwei Pills were reproduci-ble with good reproducibility,which were 0.680 4,0.450 6,0.850 8,0.676 1 and 0.757 0,the result obtained by QAMS approximated those obtained by external standard method(ESM).Conclusion:The method is simple,stable and reproducible,and can be used for the quality control of 6 alkaloid components in Zhachong Shisanwei Pills.

Objective:To establish the qualitative and quantitative methods of Lomatogonium carinthiacum(Wulf.)Reichb.in the prescription of Mongolian medicine Polypill Zhuanglun-5 decoction,and solve the phe-nomenon of Lomatogonium carinthiacum(Wulf.)Reichb.being replaced.Methods:Microscopic identification method was used to observe the pollen grains in the powder;The reference substance of swertiamarin and Lomato-gonium carinthiacum(Wulf.)Reichb.were used as the control,and the ethyl acetate methanol water formic acid(12∶2∶2∶0.5)was used as the developing agent for TLC identification;HPLC was used under the condin-tion including Agilent Eclipse Plus C18(250 mm × 4.6 mm,5 μm)as chromatographic column and 0.2％phosphoric acid solution(A)-acetonitrile(B)as mobile phase with gradient elution at 238 nm of detection wavelength were used.Contents of Zhuanglun-5 decoction from different manufacturers were determined with swertiamarin as reference substance.Results:Among the 12 batches of Zhuanglun-5 decoction,8 batches were Lomatogonium carinthiacum(Wulf.)Reichb.,2 batches were Viola yedoensis,and 2 batches were other medicinal materials.The content of swertiamarin in 8 batches of Zhuanglun-5 decoction ranged from 0.2 to 11.7 mg·g-1.Conclusion:The established identification method is simple and effective,and the content determination method is stable and has strong specificity.It can provide technical support for the supervision of the preparation,and has a reference effect for the improvement of traditional Chinese and Mongolian pharmaceuti-cal preparation standards.

The purpose of this experiment is to achieve the soluble expression of immunotoxins in Escherichia coli and avoid the complicated operation caused by the formation of inclusion bodies. An anti-mesothelin monoclonal antibody SS1 and it′s derivative SS1P which composing of SS1 and a truncated pseudomonas exotoxin PE38KDEL were used as the passenger protein. We took advantages of solubility promoting fusion tags and the self-cleaving split intein in recombinant antibody fragment and immunotoxin expression and purification. We constructed solubilizing tags-NpuCD118G fusion tags, and recombinant SS1/SS1P were fused at the C-terminal of the fusion tags. The constructs were expressed in E. coli(SHuffle T7)cytoplasm in soluble form at low temperature. Dextrin Beads 6FF column and Nickel column was used to purify the fusion protein. The self-cleavage of the fusion protein was achieved by adding the NpuN fragment. The released solubilizing tag and unreacted precursor were removed by Nickel column and the cleaved antibody fragment and immunotoxinwere further captured by Capto L. The dissociation constant of the obtained Fv and immunotoxin were determined by Fortebio. In summary, the current method could enhance the solubility of antibody fragment and immunotoxin in E. coli, and could improve the purification process. This method provides a reference for the development of a method for soluble expression and purification of immunotoxin-type pharmaceutical recombinant proteins based on the Escherichia coli expression system.

Naturally split Npu DnaE intein can mediate rapid trans-splicing and C-cleavage, which is of great use in many aspects of protein engineering. However, the degradation of NpuC during expression and purification reduces the yield and purity of recombinant protein. N2C, an extended NpuN2-containing N-terminal NpuC fragment, was constructed to improve NpuC stability. N2C was expressed in BL21(DE3) and purified by affinity chromatography. The degradation ratio was calculated by ImageJ, and the factors affecting the C-terminal cleavage reaction of intein, such as temperature, DTT concentration and N/C ratio, were also investigated. The results showed that N2C lowered the proportion of degradation to 2.7%-7.2% and the yield of C-terminal cleavage reached 90% in 30 min at 37 °C with an N/C ratio of 5∶1 catalyzed by 1 mmol/L DTT. N2C can not only improve the stability of NpuC in Escherichia coli expression system, but also retain the activity of C-terminal cleavage reaction, which is of great significance for its application in protein purification.

Intein is a functional protein that mediates self-cleavage from precursor protein and simultaneously connects the exteins on both sides of the intein via peptide bonds. Among all kinds of inteins, split intein has a wide range of applications in the field of antibody ligation. However, since the naturally split intein specifically recognizes the first three amino acid sequences “cysteine, phenylalanine, and asparagine”(CFN)of the extein, exogenous amino acids are inevitably introduced after the protein splicing reaction. In this study, the amino acid sequence “cysteine, aspartic acid, and lysine”(CDK)of the antibody hinge region was substituted for “CFN” as the recognition site for the intein and the split intein Npu DnaE was mutated into Npu*GEP DnaE. The results showed that the mutant could recognize “CDK” and the intein splicing reaction could successfully take place. The factors affecting the intein splicing reaction platform, such as pH, temperature and the concentration of NaCl and DTT were investigated in this study. The results showed that the splicing reaction of the mutant performed well, which indicated its potential usefulness in bispecific antibody assembly. In conclusion, the problem of introducing foreign amino acids was alleviated, the broadness of intein substrate was further expanded, and further technical support for the application of intein to the antibody assembly was provided.

Objective To explore the clinical value of combined detection of SCC and CEA in the diagnosis of lung cancer.Methods 382 cases from January 2015 to April 2016 in the respiratory department of our hospital were selected in this survey.Among which 172 cases with lung cancer,110cases with benign lung diseases and 100 healthy person.The CEA and SCC content in serum of all these cases were determined by chemical chemiluminescence,and the diagnostic efficacy of lung cancer by CEA or SCC alone and CEA and SCC combined examination was compared.Results In lung cancer group,the serum levels of CEA and SCC content were significantly higher than those of the normal control group and lung benign disease group(P＜0.05),and benign lung disease group were significantly higher than that of the normal control group(P＜0.05).and compared with squamous cell carcinoma and small cell lung cancer,CEA has the highest content in adenocarcinoma(P＜0.05).Meanwhile,the content of SCC in squamous cell carcinoma was obviously higher than that of small cell lung cancer and adenocarcinoma(P＜0.05),The sensitivity of CEA was significantly higher than that of SCC(P＜0.05),and the sensitivity and negative predictive value in combined detection group was significantly higher than that of CEA or SCC(P＜0.05).Conclusion SCC and CEA could be used to identify different types of lung cancer,and the combination of the two methods could improve the positive diagnosis rate,facilitate the early detection of lung cancer,early treatment,has the promotion value.

Objective To explore the experience of diagnosis and surgical treatment of carotid body tumor.Methods A retrospective analysis between November 2008 and November 2015 was proceeded,the clinical data of surgical treatment for 81 patients with carotid body tumor was collected,to analyze data by SPSS19.0,and summarize the diagnosis of carotid body tumor,choice of operation methods and curative effect and complications prevention.Results Seventy-four cases underwent surgery treatment:tumors of 52 cases were simply stripped,tumors of 13 cases were resected combined with ligation of external carotid artery.Tumors of 7 cases were resected with internal and external carotid artery ligation,3 cases of whom underwent artificial blood vessel internal carotid artery end to end anastomosis.Postoperative death in 1 case of acute myocardial infarction,complicated with cerebral infarction in 2 cases,6 cases of injury of cranial nerve relieved after symptomatic treatment.No hemiplegia,aphasia and other serious complications.Tumor size and the surgery time correlation analysis:the correlation coefficient was 0.226,no significant correlation.Conclusions CTA is the most commonly used method of preoperative examination.Surgical resection is an effective method in treatment of carotid body tumor.Prevention injury of carotid artery cr internal carotid or common carotid artery and their reconstruction is the key to a successful operation.Sufficient preoperative assessment,select the appropriate operation method,intraoperative careful performance can ensure the cerebral perfusion,is the key to prevent and reduce the complications.

Objective To understand the HIV infection status and epidemic characteristics among blood donors in Hefei area to provide the basis for recruiting the blood donors and establishing the blood detection scheme.Methods Two kinds of serological re-agent were adopted for screening the blood donors.HRV RNA in the detection samples of the nucleic acid test system and positive serological samples were simultaneously sent to the municipal Center for Disease Control for conducting the confirmation detection from 2012.Results 495 279 blood donors were performed the serological screening,44 cases were confirmed HIV positive with the detection positive rate of 0.9;112 940 samples were performed the NAT detection,20 cases were confirmed HIV positive,all were the serological dual reagent positive samples.Conclusion The HIV infection among unpaid blood donors in Hefei area shows the increasing trend year by year.Effective measures must be taken to ensure the safety of blood.

Objective To evaluate the clinical benefits and safety of endoscopic thyroidectomy via bilat -eral-nipple approach and breast approach by comparison with conventional open thyroidectomy .Methods 59 pa-tients undergoing open thyroidectomy , 76 patients undergoing endoscopic thyroidectomy via breast approach , and 122 patients undergoing endoscopic thyroidectomy via bilateral-nipple approach were retrospectively analyzed . Patients'age, operation duration, intraoperative blood loss, the first day drainage volume after operation , postop-erative hospital stay , the incidence of complications , pain visual analogue score ( VAS) , postoperative satisfaction rate as well as the evaluation of cosmetic results were compared among different groups .Results No significant difference was observed in the intraoperative blood loss and the postoperative hospital stay among different groups . The operation duration and the first day drainage volume after operation in the endoscopic thyroidectomy groups ((103.5 ±26.2)min vs (108.1 ±23.9)min,(106.5 ±31.0)ml vs (117.4 ±38.8)ml)were significantly high-er than those in the open thyroidectomy group((65.3 ±22.4)min and (50.5 ±23.8)ml)(P<0.05, respective-ly).No significant difference was observed in the complication incidence among different groups .On the first day after operation, VAS of the endoscopic thyroidectomy groups (2.8 ±0.4 vs 2.9 ±0.4)was significantly lower than that of the open thyroidectomy group(3.8 ±0.6)(P<0.05, respectively), even though on the second and the third day no significant difference was observed among different groups .Besides, postoperative satisfaction rate and the evaluation of cosmetic results in endoscopic thyroidectomy groups were significantly higher than those in the open group(P<0.05, respectively).Furthermore, 90 days after operation, the satisfaction rate and the eval-uation of cosmetic results in the endoscopic thyroidectomy group through bilateral -nipple approach (96.7%,8.8 ± 0.9)were significantly higher than those in the endoscopic thyroidectomy group through breast approach (92.1%, 7.3 ±0.7)(P<0.05, respectively).Conclusions Endoscopic thyroidectomy is a feasible and safe procedure . Endoscopic thyroidectomy through bilateral-nipple approach has a higher long-term satisfaction rate as well as bet-ter cosmetic results , which is an ideal choice of thyroidectomy .