Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation / 生物工程学报

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

The I226R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immune response / 生物工程学报

This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.

Establishment of a candidate reference method for angiotensin Ⅱ in human plasma / 中华检验医学杂志

Objective:To establish a candidate reference method for the determination of angiotensin Ⅱ in human plasma by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and to evaluate its performance.Methods:Using [ 13C 6- 15N]-angiotensin Ⅱ as the internal standard, the plasma was accurately weighed by gravimetric method and mixed with a certain amount of internal standard. At the same time, enzyme inhibitor was added. After zinc sulfate solution protein precipitation and reversed-phase solid-phase extraction plate treatment, it was analyzed by liquid chromatography tandem mass spectrometry. The multi reaction ion monitoring mode(MRM)was selected by mass spectrometry to detect specific ion fragments of angiotensin Ⅱ and internal standard. The linearity, sensitivity, precision, recovery rate and uncertainty of the performance of the established method were evaluated according to ISO15193. Results:Angiotensin Ⅱ had good linearity in the range of 10-1 000 pg/g ( r=0.999 5), the lower limit of quantification was 7.68 pg/g, the analytical recoveries were 97.14% to 102.85%, intra-batch imprecision≤3.21%, inter-batch imprecision≤2.96%, and total imprecision≤3.67%. Conclusion:A method for the determination of plasma angiotensin Ⅱ was established by ID-LC-MS/MS. The method is accurate and reliable, and is expected to be a reference method for the determination of plasma angiotensin Ⅱ.

Study on plasmid-mediated blaNDM-1 gene in carbapenem-resistant Citrobacter freundii / 中国感染与化疗杂志

Objective To investigate the transmission of blaNDM-1 gene in carbapenem-resistant Citrobacter freundii. Methods A total of 18 strains of NDM-1-producing C. freundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 to October 2014. The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System. Conjugation experiments, pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to determine the transferability of plasmids. Results The antibiotic susceptibility results showed that all the NDM-1-producing C. freundii isolates were resistant to penicillins, cephalosporins and carbapenems. All isolates exhibited different level resistance to other antibiotics. Conjugation experiments revealed that the plasmids harboring blaNDM-1 in 13 strains were transformed into E. coli 600, and exhibited carbapenem resistance. PFGE and Southern blot hybridization found that blaNDM-1 was located on a 33.3 kb plasmid in 16 isolates and on 33.3-54.7 kb plasmid in 2 isolates. Conclusions Our findings suggest that plasmids contribute to the horizontal dissemination of blaNDM-1 gene in carbapenemresistant C. freundii.

The clinical significance of rheumatoid arthritis merger antinuclear antibody positive / 国际检验医学杂志

Objective To understand the fluorescence pattern of antinuclear antibody(ANA)and the distribution of target anti-gen and ANA′s impact on rheumatoid arthritis(RA)RA.Methods The data of rheumatoid factor(RF)and anti-cyclic citrullinated peptide antibodies(anti-CCP antibodies)and ANA of 860 RA patients during 2014-2015 were collected and divided into 2 groups according to the ANA levels.The differences of RF and anti-CCP antibodies level between 2 groups were analyzed.Analyzed the fluorescence pattern of RA merger ANA and the distribution of target antigen by statistical.Results Of the 860 RA patients,the positive rate of ANA was 37.6%,the major fluorescence pattern of RA merger ANA was speckled pattern,anti SSA positive rate was 21.7%.The RF and anti-CCP antibodies level had statistical difference between ANA positive group and ANA negative group (P <0.05).Conclusion RA merger ANA positive can show different fluorescence patterns and target antigen,the speckled pattern and anti-SSA positive are the main pattern.The severity of arthritis symptoms and the degree of bone destruction between ANA positive group and ANA negative group were difference.Anti-ANA positive has a certain promoting effect on the progression of RA disease.

Study on relationship between human papilloma virus,telomerase gene and chromosome 3 number with cervical lesions / 国际检验医学杂志

Objective To explore the relationship between the expression of human telomerase RNA component (TERC) gene , human papilloma virus (HPV) infection and mutation of chromosome 3 number with cervical lesions .Methods 81 women received the treatment in the Gynecology Department of the Second Affiliated Hospital of Kunming Medical University from June 2008 to February 2009 ,including the healthy group(normal pathological examination ,20 cases) ,CIN1 group(28 cases) ,CIN2 group(12 ca‐ses) ,CIN3 group(9 cases) and cervical cancer group(12 cases) .The TERC gene expression in uterine epithelial exfoliated cells was detected by using the fluorescence in situ hybridization (FISH) method ,meanwhile the HPV infection was detected by using the real time fluorescence quantitative polymerase chain reaction (FQPCR) technology .The correlation between cervical cancer with TERC gene and HPV was analyzed .At the same time the number of chromosome 3 mutations in 81 cases was recorded .Results In the cervical lesion detection ,the detection positive rate had no statistical difference between the TERC gene detection and HPV detec ‐tion (P> 0 .05) ,their positive rates in the CIN 1 ,CIN2 ,CIN3 and cervical cancer groups were significantly higher than that in the healthy group (P 0 .05) , while between the CIN3 group and the cervical cancer group had statistical significance (P< 0 .05) ,the higher the malignant degree , the higher the positive rate .The abnormal mutation rate of chromosome 3 number was 0% in the healthy group and the CIN1 group ,16 .7% in the CIN2 group ,66 .7% in the CIN3 group and 100 .0% in the cervical cancer group ,the positive rate in the CIN3 group and the cervical cancer group was significantly higher than that in the healthy group ,CIN1 group and CIN2 group ,the differ‐ences were statistically significant (P< 0 .05) .Conclusion The TERC abnormal gene expression ,high risk HPV infection and mutation of chromosome 3 number could play an important synergistic effect during the process of occurrence and progression of cervical cancer .