The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.

Objective:To investigate the immunogeneicity of a subunit vaccine of capsid protein precursor(P1) of swine vesicular diseas(SVD).Methods:In this study,the guinea pigs were immunized with the home-made antigen,T-lymphocyte proliferation response,blocking ELISA and micro-neutralization assay were used to detect the effect of the immunized responses in guinea pigs.Results:The results indicated that a retroviral-based vaccine carrying the capsid protein precursor(P1) of SVD was able to elicit strong SVDV-specific humoral immune responses in guinea pigs.Conclusion:It encourages further work towards the development of a vaccine against SVDV infection.

Taking the Department of Obstetrics and Gynecology as an example, this paper expounds a series of practices of online education, in the process from pre-course training, platform selection, curriculum design, to student evaluation, etc. From that, we can provide a reference for carrying out online teaching in response to the epidemic for clinical medical colleges which have not been exposed to the online course. Furthermore, it is a new attempt to explore a way to make a change of the traditional teaching method and deepen the reform of clinical teaching in our hospital after the epidemic, not only in professional medical education, but also in the popularization of medical knowledge.

Objective:To investigate the early clinical effects of orthopedic surgery robot-assisted double Endobutton titanium plate internal fixation in the treatment of fresh acromioclavicular joint dislocation.Methods:Thirty-nine patients with fresh acromioclavicular joint dislocation were included from January 2020 to January 2022. A total of 19 patients were treated with double Endobutton suspension internal fixation assisted by the domestic third-generation orthopaedic surgical robot (TiRobot ? 2.0) Dimensity system. There were Rockwood type III in 11 cases, type IV in 8 cases. Twenty cases were treated with conventional incision double Endobutton internal fixation, with Rockwood type III in 13 cases, type V in 7 cases. The operation duration, blood loss volume, incision length and hospitalization time were compared between the two groups. The following CT parameters of acromioclavicular joint at 2 days and 1 year after operation, distance between distal inferior cortex of clavicle and subacromial cortex, distance between upper and lower endobuttons, horizontal distance between anterior edge of distal clavicle and anterior edge of acromion and diameter of coracoid process and diameter of clavicular tunnel were measured. The visual analogue score (VAS), Constant-Murley shoulder function score and shoulder abduction activity were also evaluated before and at 12 months after operation. Results:The follow-up duration was 10.8±2.4 months in the robot group and 11.5±3.1 months in the routine group. The VAS score of the robot group decreased from 5.3±2.1 to 0.3±0.2 at 12 months after operation ( t=10.46, P=0.014). The Constant-Murley score increased from 55.6±6.4 to 92.0±4.2. The range of shoulder abduction increased from 42.2°±5.4° to 172.6°±6.1° ( t=17.24, P<0.001). The operation duation of the robot group was 74.4±6.6 min, which was longer than that of the conventional group 61.7±7.2 min ( t=5.43, P=0.037). There was no significant difference in VAS score, Constant-Murley score, shoulder abduction activity or CT measurement between the two groups ( P>0.05). During the follow-up, two cases in the robot group had cortical osteolysis on the supraclavicular surface, one case in the conventional group had loss of reduction, one case in the supraclavicular cortical osteolysis, and 4 cases in the cortical defect on the side of the coracoid process tunnel. Conclusion:Orthopedic robot-assisted and conventional incision with double Endobutton titanium plate internal fixation in treating fresh acromioclavicular joint dislocation can achieve satisfied early clinical effects. Accurate establishment of clavicle and coracoid bone tunnel assisted by robot can overcome the defects of bone tunnel deviation in conventional incision operation and can prevent reduction and bone loss. However, robot-assisted and conventional incision Endobutton internal fixation could enlarge bone tunnel.