Lyophilized mRNA-lipid nanoparticles vaccine with long-term stability and high antigenicity against SARS-CoV-2

Advanced mRNA vaccines play vital roles against SARS-CoV-2. However, due to their poor stability, most current mRNA delivery platforms need to be stored at -20{degrees}C or -70{degrees}C, which severely limits their distribution. Herein, we present lyophilized SARS-CoV-2 mRNA-lipid nanoparticle vaccines, which can be stored at room temperature with long-term thermostability. In the in vivo Delta virus challenge experiment, lyophilized Delta variant mRNA vaccine successfully protected mice from infection and cleared the virus. Lyophilized omicron mRNA vaccine enabled to elicit both potent humoral and cellular immunity. In booster immunization experiments in mice and old monkeys, lyophilized omicron mRNA vaccine could effectively increase the titers of neutralizing antibodies against wild-type coronavirus and omicron variants. In humans, lyophilized omicron mRNA vaccine as a booster shot could also engender excellent immunity and had less severe adverse events. This lyophilization platform overcomes the instability of mRNA vaccines without affecting their bioactivity, and significantly improved their accessibility, particularly in remote regions.

Protein kinases in cardiovascular diseases / 中华医学杂志(英文版)

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Therefore, exploring the mechanism of CVDs and critical regulatory factors is of great significance for promoting heart repair, reversing cardiac remodeling, and reducing adverse cardiovascular events. Recently, significant progress has been made in understanding the function of protein kinases and their interactions with other regulatory proteins in myocardial biology. Protein kinases are positioned as critical regulators at the intersection of multiple signals and coordinate nearly every aspect of myocardial responses, regulating contractility, metabolism, transcription, and cellular death. Equally, reconstructing the disrupted protein kinases regulatory network will help reverse pathological progress and stimulate cardiac repair. This review summarizes recent researches concerning the function of protein kinases in CVDs, discusses their promising clinical applications, and explores potential targets for future treatments.

Introduction of papillomavirus rapid interface for modelling and economics (PRIME) and an example of China′s data analysis / 中华预防医学杂志

Cervical cancer has become an important disease that jeopardizes women′s health, causing hundreds of thousands of new cases annually. Human papillomavirus (HPV) is the leading cause for cervical cancer. Since the world′s first HPV vaccine was licensed in 2006, 92 countries around the world have introduced them in national immunization programs. The WHO recommends that scientific economic evaluation should be achieved before the introduction, but this is more difficult for low-and middle-income countries. Therefore, this article introduces a mathematical model recommended by WHO in 2014 to quickly and easily accomplish economic evaluation of HPV vaccine -the PRIME, and take the China′s 2018 data published by International Agency for Research on Cancer (IARC) as an example. The evaluation result shows that the introduction of HPV vaccine in Chinese 12-year-old women is cost-effective.

White-Matter Hyperintensities and Lacunar Infarcts Are Associated with an Increased Risk of Alzheimer's Disease in the Elderly in China

BACKGROUND AND PURPOSE: This study investigated the contribution of white-matter hyperintensities (WMH) and lacunar infarcts (LI) to the risk of Alzheimer's disease (AD) in an elderly cohort in China. METHODS: Older adults who were initially cognitively normal were examined with MRI at baseline, and followed for 5 years. WMH were classified as mild, moderate, or severe, and LI were classified into a few LI (1 to 3) or many LI (≥4). Cognitive function was assessed using the Mini Mental State Examination and the Activities of Daily Living scale. RESULTS: Among the 2,626 subjects, 357 developed AD by the end of the 5-year follow-up period. After adjusting for age and other potential confounders, having only WMH, having only LI, and having both WMH and LI were associated with an increased risk of developing AD compared with having neither WMH nor LI. Moderate and severe WMH were associated with an increased risk of developing AD compared with no WMH. Furthermore, patients with many LI had an increased risk of developing AD compared with no LI. CONCLUSIONS: Having moderate or severe WMH and many LI were associated with an increased risk of developing AD, with this being particularly striking when both WMH and LI were present.

A case-control study on risk factors for intrauterine HBV infection / 重庆医学

Objective To explore the risk factors for hepatitis B virus (HBV) intrauterine infection to provide a scientific ev idence for itsprevention.Methods Three hundred and twelve pregnant women of HBsAg positive screened from April 2013 to May 2015 served as the research subjects and were followed up until 6 months after birth.The infantile mothers of HBsAg and/or HBV DNA positive were selected as the intrauterine infection case group,while other mothers served as the control group.The Logistic regression analysis was adopted to analyze the risk factors for intrauterine HBV infection.The questionnaire survey method was used to collect the basic data and time-resolved immunofluorescence assay was used to detect HBsAg.PCR was adopted to measure level of HBV DNA and automatic biochemistry analyzer was used to measure the hepatic functional parameters including ALT,AST,triglyceride and cholesterol.Results The single factor analysis results indicated that HBeAg,HBV DNA,contamination of amniotic fluid and sexual behavior during pregnancy were related to HBV intrauterine infection(P＜0.05).The multiple variate Logistic regression results showed that positive HBeAg(OR=2.76,95 ％ CI=1.19-7.94),positive HBV DNA(OR=9.62,95 ％ CI=2.58-35.33),and sexual behaviors during pregnancy (OR =1.53,95 ％ CI =1.07-6.40) were the risk factors for intrauterine HBV infection.Conclusion Pregnant women with positive HBeAg,positive HBV DNA and sexual behavior during pregnancy may be the high risk factors for neonatal intrauterine HBV infection.

Role of immune-related GTPase M I in cortical neurons autophagy of mice with sepsis-induced brain injury / 中南大学学报(医学版)

Objective:To investigate the role of immune-related GTPase M1 (IRGM1) in cortical neurons autophagy in mice with sepsis induced brain injury (SIBI).Methods:Sixty wild-type C57BL/6 mice and sixty IRGM1 gene knockout C57BL/6 mice were randomly divided into 4 groups:a sham-operated wild-type (SWT) group,a cecal ligation and puncture (CLP) model wild-type (MWT) group,a sham-operated knockout (SKO) group,and a CLP model knockout (MKO) group.Models of mice with sepsis were established by CLP.Six hours of after CLP,the neurobehavioral scores for mice were recorded.The mice were diagnosed with SIBI and enrolled for the studies in next step if the neurobehavioral score was less than 6 in the MWT and MKO groups.The sham operation group only opened the abdominal cavity without CLP.Pathological changes in mouse cerebral cortex were observed by HE staining.Electron microscope was used to observe the ultrastructure of autophagy in cortical neurons.The expression of IRGM1 and INF-γ mRNA in the cerebral cortex of mice were detected by Real time quantitative PCR.The protein expression of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ,LC3-Ⅰ,sequestosome-1 (SQSTM1) and IRGM1 were measured by Western blot.Immunofluorescence staining was used to examine the expression of IRGM 1 in mouse cortical neurons.Results:In the MWT group,the cortical neurons showed dilated endoplasmic reticulum,swelling mitochondria,and increased number of autophagosomes after 6 or 24 h of CLP in contrast to the SWT group.At 6 h after CLP,the expression of LC3-Ⅱ in the cerebral cortex began to up-regulate,and the up-regulation was maintained till 96 h after CLP;on the contrary,SQSTM1 began to decline after 6 h of CLP.Compared with SWT group,IRGM1 was strongly up-regulated in the cerebral cortex of mice at both mRNA and protein levels in the MWT group after 12 h of CLP,and the mRNA expression of IFN-γ was also increased significantly (P＜0.05).At 24 h after CLP,the IRGM1 expression of cortical neurons in the MWT group was significantly higher than that in the SWT group.The baseline of autophagy activity was quite low in the cerebral cortex cells in the SWT and the SKO groups.There was almost no detected expression of LC3-Ⅱ;conversely,the expression of SQSTM1 was very high after 12 h of CLP.However,the expression of LC3-Ⅱ was significantly up-regulated and the expression of SQSTM1 was down-regulated in the MWT group (P＜0.05).On the other hand,there was almost no detected LC3-Ⅱ expression in cerebral cortex in the MKO group,and the expression of SQSTMI was up-regulated.At 6 h after CLP,the incidence of SIBI was 90％ (27/30) in the MWT group,and 96.67％ (29/30) in the MKO group.At 12 h of CLP,the neurobehavioral scores in the MKO group was significantly lower than that in the MWT group (4.97±0.71 vs 5.43±0.86;t=2.284,P=0.026).HE staining showed that mice in the MKO group suffered severe cerebral cortex injury,and the number of nerve cells was significantly reduced compared with that in the MWT group.Conclusion:The IRGM1 exerts a protective effect on the brain of the mice with SIBI,and its mechanism might be related to the regulation of autophagy in mouse cortical neurons.

Autophagy in hippocampal nerve cells from rats with sepsis-associated encephalopathy / 中南大学学报(医学版)

OBJECTIVE@#To show evidence of the autophagy in hippocampal nerve cells from rats with sepsis-associated encephalopathy (SAE) in vivo and to investigate the expression of microtubule-associated protein 1 light chain 3 (LC3). 
@*METHODS@#A rat model of sepsis was established by the cecal ligation and puncture (CLP). A total of 60 male Wistar rats (30 days old) were randomly divided into a sham group (n=10) and a CLP group (n=50). At 12 hours after CLP, the electroencephalogram (EEG) and somatosensory evoked potential (SEP) changes in rats were monitored and the neurobehavioral score was measured. According to the occurrence of SAE, the CLP group was further divided into an SAE(+) group and an SAE(-) group. Histopathological changes in hippocampus were observed by hematoxylin-eosin staining. An electron microscope was used to observe autophagosome formation and lysosome activation in the hippocampal nerve cells. Expressions of LC3-I and LC3-II protein were measured by Western blot. 
@*RESULTS@#Five of 50 rats in CLP group died in 12 hours after CLP. According to the low neurobehavioral score and abnormal EEG and SEP, 16 rats were diagnosed as SAE. The incidence of SAE was 35.56% (16/45). Compared with the sham group or the SAE(-) group, the frequency of α wave in SAE(+) group was significantly decreased at 12 hours after CLP, the δ wave increased, the P1 amplitude decreased, and the latency of SEP waves (P1 and N1) was prolonged (P<0.05). The morphology of hippocampal nerve cells was obvious in a status of edema. Pyramidal cells decreased significantly, even dissolved, and cell arrangement was in disorder in the SAE(+) group. But these cells were normal in the sham group and the SAE(-) group. The structure of hippocampal nerve cells was disordered, and the autophagy, granular matrix and square or rectangular crystals were found in the SAE(+) group. However, there was no autophagy both in the sham group and the SAE(-) group. LC3-II/LC3-I ratio in the hippocampal nerve cells was increased significantly at 12 hours after CLP in the SAE(+) group when compared with that in the sham or the SAE(-) group (P<0.05). 
@*CONCLUSION@#There is autophagy in hippocampal nerve cells from rats with SAE and the LC3-II/LC3-I ratio is increased significantly.

Effect of maternal deprivation on learning and memory ability and hippocampal nerve regeneration in rats with hypoxic-ischemic brain damage / 中华实用儿科临床杂志

Objective To observe the effect of maternal deprivation(MD)on learning and memory ability and hippocampal pathology and nestin expression in rats with hypoxic -ischemic brain damage (HIBD).Methods The models of HIBD SD male rats were established by the method of Rice,and were randomly divided into 2 groups:MD group and control group.In addition,the sham -operation group(sham group)models were established.The MD group rats were separated from their mother 3 hours per day from the second day after modeling to the 21 st postnatal day.After 28 postnatal days,Morris water maze was used to evaluate the learning and memory ability of the rat models.HE stai-ning was employed to observe the hippocampal pathological change in the rats.Then,the expression of nestin in the hip-pocampus was measured by the method of immunohistochemistry.Results Their body mass changes showed that quali-ty of sham group was higher than that of the control and the MD groups,and quality was improved in the control group, compared with the MD group,and the differences were statistically significant(q =9.860 8,3.880 7,5.980 1 ,all P <0.05).The water maze scores of the MD group in place navigation test and spatial probe test were much lower than that of the control group and the sham group,and the scores of the sham group were higher than that of the control group,the differences were statistically significant(all P <0.05 ).The findings of HE stain showed that the pathology in the right -sided hippocampus of the sham group was normal and neurons were well -arranged,and that of control group was minimally abnormal,and the neurons were almost arranged orderly and remained normal.While,the pathomorpholo-gy of the MD group was obviously abnormal,the neurons were arranged disorderly,many of the neurons lost.According to the immunohistochemical findings,the number of nestin -positive cells in right -sided hippocampus of the MD group was significantly less than that of the control group,and the number of nestin -positive cells of the sham group was less than that of the MD group,which showed significant differences among the groups(all P <0.05).Conclusions MD aggravated injury to learning and memory ability of neonatal rats with HIBD,and decrease the number of nestin -posi-tive cells of MD markedly,which is not good for the recovery of brain injury.

Effect of Dexamethasone on LC3 expression of neurons in cerebral cortex of juvenile rats with sepsis / 中华实用儿科临床杂志

Objective To investigate the effect of Dexamethasone on microtubule - associated protein 1 light chain 3(LC3)expression of cells and neurons in cerebral cortex of juvenile Wistar rats with sepsis. Methods Models of juvenile Wistar rats with sepsis were established through cecal ligation and puncture(CLP). Totally 60 cases of 30 -day - old juvenile male Wistar rats were randomly divided into sham - operation group(10 cases),non - treated group (25 cases)and Dexamethasone group(25 cases). Twelve hours after CLP,rats in Dexamethasone group were injected with Dexamethasone(1 mg / kg)via tail vein every other day,with a total of 3 times. The same dose of saline was used in the non - treated group. All rats were killed at the age of 40 days. Expressions of LC3 and neuronal nuclei(NeuN)of cells in cerebral cortex of rats were detected by using immunofluorescence assay,and the number of positive cells was calculated by using image analysis system software. Expressions of LC3 - Ⅰ and LC3 - Ⅱ protein were measured by a-dopting Western blot. Results Three hours after CLP,rats appeared to be curled up and showed piloerection and shi-vering and the neurobehavioral score in non - treated group was significantly lower than that in sham - operation group (t = 9. 895,P = 0. 000). Twelve of 25 rats in Dexamethasone group died in 10 days after CLP(48% ),while 8 of 25 rats in non - treated group died(32% ),and the difference was not statistically significant between the 2 groups(χ2 =1. 333,P = 0. 248). The immunofluorescence staining and image analysis showed the percentage of LC3 positive cells in non - treated group was significantly increased(0. 606 7 ± 0. 030 1 vs 0. 353 3 ± 0. 025 8,t = 15. 644,P = 0. 000;0. 606 7 ± 0. 030 1 vs 0. 270 3 ± 0. 019 4,t = 22. 450,P = 0. 000). In non - treated group,the LC3 expression of cells in the cerebral cortex of rats was up - regulated,and the LC3 - Ⅱ/ LC3 - Ⅰ ratio was significantly higher than that in sham operation group and Dexamethasone group(0. 413 3 ± 0. 022 5 vs 0. 205 0 ± 0. 015 2,t = 18. 802,P = 0. 000;0. 413 3 ± 0. 022 5 vs 0. 185 0 ± 0. 023 5,t = 17. 206,P = 0. 000). The LC3 positive neurons in the cerebral cortex of rats were less in sham operation group and Dexamethasone group. The LC3 positive neurons were more in non - treated group than that in sham operation group and Dexamethasone group(0. 580 0 ± 0. 020 0 vs 0. 298 3 ± 0. 014 7,t =27. 783;P = 0. 000;0. 580 0 ± 0. 020 0 vs 0. 261 7 ± 0. 017 2,t = 28. 614;P = 0. 000). Conclusions The LC3 expres-sion of cells in the cerebral cortex of juvenile Wistar rats with sepsis was up - regulated,LC3 - Ⅱ/ LC3 - Ⅰ ratio in-creased,and the number of LC3 positive neurons also increased,while Dexamethasone could have inhibitory effect on them.

Drug resistance situation in 2 271 tuberculous patients and epidemiological characteristics analysis / 重庆医学

Objective To investigate the drug resistance situation and the epidemiological characteristicin the tuberculoupatientin ordeto provide the reference foeffective prevention and treatmenof T.MethodTotally 2 271 patientwith posi-tive sputum smeapulmonary tuberculosi(TB) in the Jiangbei Branch Hospital of Chongqing Zhongshan Hospital from January 2009 to Decembe2013 were performed the drug resistance detection of 10 kindof anti-Tdrugs(isoniazid ,streptomycin ,rifampi-cin ,ethambutol ,para-aminosailcyliacid ,kanamycin ,pasiniazid ,protionamide ,capreomycin ,and ofloxacin) .ResultOf all 2 271 ca-seof positive sputum culture pulmonary T,the total drug resistance rate and total multi-drug resistance rate were 61 .91% and 26 .29% ,respectively ;the total drug resistance ratein initial treatmenand retreatmenpatientwere 52 .85% and 78 .06% respec-tively ,and the multi-drug resistance ratewere 16 .70% and 43 .38% ,respectively ;the top five in the average drug resistance ratefrom high to low were streptomycin(39 .45% ) ,isoniazid(33 .20% ) ,rifampicin(32 .94% ) ,para-aminosailcyliacid(31 .40% ) and pasiniazid(21 .53% );the anti-Ttreatmenhistory ,gende,age and occupation were significantly associated with the drug resist-ance occurrence (P＜0 .05) .Conclusion The drug resistance situation of the Tpatientin the local areof Chongqing iseriouin the recen5 year.The standardized managemenand treatmenon the Tpatientwith drug resistance should be strengthened .

Effect and mechanism of xCT on invasion and migration of nasopharyngeal carcinoma cells in vitro / 中国医师杂志

Objective To explore the role of xCT in the metastasis and drug resistance of nasopharyngeal carcinoma (NPC),to provide new evidences for the mechanism of NPC metastasis and drug resistance,and to enhance effects of chemotherapy.Methods Fluorescence quantitative polymerase chain reaction (PCR) and Western blot were used to detect the xCT expression of 5-8F and 6-10B.The xCT eukaryotic expression vector was constructed to transient transfect 6-10B cell lines.The 5-8F cell lines were treated with xCT antisense oligodeoxynucleotide (ASO).Scratching assay and transwell method were used to detect the cell metastasis and invasion abilities.The expressions of matrix metalloproteinase 1 (MMP1) of all the cell lines were surveyed by Western blot.Results The results of fluorescence quantitative PCR and Western blot showed that the expression of xCT in 5-8F was higher than 6-10B in the levels of mRNA and protein.Exogenous overexpression of xCT enhanced metastasis and invasion abilities of 6-10B cells.Silencing and inhibition of xCT could decrease metastasis and invasion abilities of 5-8F cells.The expressions of MMP1 protein in 5-8F were higher than 6-10B,and they were positively correlated with the expression of xCT.Conclusions xCT is closely related to the metastasis and invasion abilities of nasopharyngeal carcinoma.xCT could enhance the metastasis and invasion abilities of NPC cells.xCT might mediate proliferation and matastasis/invasion of NPC cells through MMP1.

A Processing Method of EEG Signals Based on Second Generation Wavelet Transform and Blind Signal Separation / 航天医学与医学工程

Objective To study a processing method for EEG signals mixed with EOG and ECG signals disturbance.Methods First,the EEG was denoised by the hard threshold method,the soft threshold method,the compromise threshold method and the ? law threshold method in the second generation wavelet,and then the denoised EEG which still contained EOG and ECG was separated by fast independent component analysis( FastICA) algorithm.Results The ? law threshold method of the second generation wavelet had better denoising effect and FastICA algorithm had more ideal separate performance.Conclusion It is an effective preprocessing method for EEG in denoising with the ? law threshold method of the second generation wavelet and then in separating disturbance of independent source with FastICA algorithm.

Disinfection efficacy of SZ herbal disinfectant on bacterium and virus in suspension / 第三军医大学学报

Objective To determine the main components of SZ herbal disinfectant and to observe its inactivation efficiency on microorganism in suspension. Methods The spectrophotometer examination, neutralizer screening trial, bacteria count trial, phage plaque count trial and cells infection test had been used to determine contents of ursolic acid in SZ herbal disinfectant and the disinfection efficacy. Results The ursolic acid contents in original SZ disinfectant reached 436.43 ?g/ml and decreased by 8.07% after a year of deposit. Treated by disinfectant for 5 min and 1∶5 dilution solution for 10 min, the Staphylococcus aureus (SA) and Escherichia coli (EC) in suspension could be killed with the efficiency of 100%. Treated with SZ disinfectant and the dilution solution (1∶50) respectively, the Candida albicans could be killed with the efficiency of 90.97% and 51.10%. Treated with the dilution solution for 3 min, the f2 bacteriophage and PV could be reduced by 100% and 9.00 log. Conclusion With the main component of ursolic acid, the SZ herbal disinfectant is a kind of natural disinfectant. It has a satisfactory inactivation efficacy on bacterial vegetative form and virus.

Treatment of avascular necrosis of femoral head by implanting a composite of autogenous bone marrow and noncelluar tissue engineered bone allograft with BMP / 中华骨科杂志

Objective To investigate the effect of treatment of avascular necrosis of femoral head in adolescence by implanting a composite of autogenous bone marrow, bone morphogenetic protein(BMP), and noncelluar tissue engineered bone allograft. Methods The BMP was partially purified from bovine cortical bone (bBMP) by Urist method. The bone allograft chips were mixed with bBMP and bone marrow. The composite was implanted into the necrotic area of the femoral head after core decompression, then partial deminerallized allograft fibula segments were inserted in the core decompression holes to support the necrotic area in preventing the collapse in 64 adolescent patients (78 hips). Results 55 cases (67 hips) were followed up for 3 months to 6 years(mean 44 months). 28 case(36 hips) in FicatⅠ,Ⅱ were followed up over 3 years, of whom there were no obvious pain and dysfunction in 18 cases(22 hips), high density new bone was shown in core decompression area in CT scanning, and no evidence of progressive necrosis. There was no worsening of the symptoms in 4 cases(6 hips) in Ficat Ⅰand Ⅱ, but the lesion progressed. In 6 cases (8 hips) in Ficat Ⅲ, there were no obvious pain and dysfunction in 2 cases, but 4 cases underwent total hip replacement because of persistent pain and progressive lesion. Conclusion The partial demineralized allograft fibula can provide direct mechanical support to prevent the necrotic femoral head from progress and collapse, autogenous bone marrow and BMP is able to promote new bone formation. The method can be used as an alternative for the treatment of osteonecrosis of femoral head at stage Ⅰ,Ⅱ.

Epidemiological investigation of host and focus of natural infection on hemorrhagic fever with renal syndrome in migration areas of the Yangtze River Three-Gorge Reservoir Chongqing region / 第三军医大学学报

Objective　To ascertain the natural infection rate of hemorrhagic fever with renal syndrome virus (HFRSV) among its hosts and the type of the natural foci for providing some baseline data for the immigrant health and epidemic prevention of the Three-Gorge region. Methods　Epidemiological survey on the field was performed including epidemiological data collection, ecology of rodents and pathogen detection. HFRS virus antigen of hosts were detected by the direct immunofluorescent assay (DIFA) technique and determination of HFRSV-RNA by ISH were carried out from HFRSV-Ag-positive animals. Results　HFRSV-Ag-positive animals were found in 5 migration areas ie Baitao Town of Fuling Section, Wansheng Village of Fengjie County and Dachang Town of Wushan County. The positive hosts were as follows, Rattus Norvegicus, Apodemus agrarius, Anourusurex squamipes, Mus musculus and Rattus flavipectus. The positive rate of HFRSV in the mice of 5 migration areas were 19.4%, 17.0%, 14.0%, 13.7%, and 8.5% respectively. The results showed that the lung tissues of some hosts in all five migration areas were HFRSV-RNA-positive. Baitao Town and Peishi Town were attributed to mixture type epidemic areas while. Kangle Town, Wansheng Village and Dachang Town were domestic rats type epidemic areas. Conclusion　This study shows that the five migration areas are natural epidemic foci of HFRS. It is predicted that maximum risk of HFRS breakout or epidemic may take place after the completion of the San Xia Reservoir(the Three-Gorges Reservoir), which results from rodent moving toward higher land. Therefore, deratization and preventive measures for rat are important in migration areas.

Construction and identification of expression of superantigen SED mutants / 第三军医大学学报

Objective To construct staphylococcal enterotoxin D (SED) mutants expressed in Escherichia coli ( E. coli ). Methods The expected mutants were introduced into the SED DNA by megaprimer PCR method. The PCR products ligated to plasmid pTrcHis B were transformed into E. coli DH5? for IPTG induced expression. The target protein was purified by Ni NTA metal affinity chromatography and analyzed by SDS PAGE and immunoblotting. Results The sequencing results showed that mutant nucleic acids were successfully introduced at the expected sites of SED gene. SDS PAGE and immunoblotting confirmed that the proteins of SED mutants were obtained by Ni NTA metal affinity chromatography. The mutants were named as SEDN23A, SEDN23A/H26R, SEDF45A, SEDL59A, SEDN61A, SEDI92A, and SEDF203A, respectively. Conclusion Several SED mutants are successfully constructed, which lays a foundation for subsequent studies of immune recognition of SED.