Preparation and identification of rabbit anti-marmoset IgG-HRP conjugate antibody / 中国比较医学杂志

Objective To purify marmoset serum IgG, prepare and identify the antiserum and the rabbit anti-marmoset antibody IgG-HRP (horseradish peroxidase). Methods Using SDS-PAGE analysis to identify the serum IgG from HiTrapTM Protein G. The antiserum titer was determined by double immunodiffusion assay. The rabbit anti-marmoset IgG was labeled with HRP by improved sodium periodate method. ELISA and western blotting were used to evaluate the concentration and specificity of rabbit anti-marmoset IgG-HRP. Results The purity of purified marmoset serum IgG determined by SDS-PAGE was higher than 95% , and the anti-serum titer of the anti-marmoset IgG polyclonal antibody was 1∶64. The concentration of rabbit anti-marmoset IgG-HRP identified by direct ELISA was 1∶256 000, and that by western-blotting was 1∶15 000, with a strong specificity. Conclusions The IgG-HRP marker antibody is prepared and the specificity and concentration are identified by ELISA and western blotting. It reserves the resources for the detection system of marmoset pathogens and the molecular immunological testing system.

Microsatellite marker design and genetic analysis for the common marmoset / 中国比较医学杂志

Objective To screen and optimize the microsatellite DNA primers of the laboratory common marmoset, analyze and evaluate the population genetic quality for the marmosets (Callithrix jacchus) introduced into the Institute of Medical Laboratory Animal science, Chinese Academy of Medical Sciences. Methods A total of 30 marmosets were randomly chosen, and their genome DNA from blood was extracted using phenol/chloroform method. The microsatellite DNA was amplified using standard polymerase chain reaction (PCR). The amplification products were tested by STR scanning after 2% agrose gel and 8% PAGE electrophoresis. The data processing and genetic analysis were completed using the Popgene1. 32 software. Results A total of 20 pairs of microsatellite loci showed genetic polymorphism, and 147 alleles were detected. The number of allele was 5 to 10, average 7. 35. The effective allele was 2. 2500 to 6. 3830, average 4. 0402. The observed heterozygosity was 0. 000 to 0. 4667, average 0. 1533. The expected heterozygosity was 0. 1424 to 0. 4350, average 0. 2506. The Shannon diversity index was 1. 2242 to 2. 0324, average 1. 5949. The polymorphic information content was 0. 5366 to 0. 8254, average 0. 7053. Conclusions The 20 pairs of marmoset microsatellite primers are genetically highly diverse and are in a Hardy-Weinberg equilibrium.

Effects of transport and storage conditions on the nucleic acid detection for three kinds of mouse RNA viral samples / 中国比较医学杂志

Objective To analyze the effect of transport and storage conditions on the detection of pathogenic nucleic acid MHV, Reo-3, MNV in laboratory mouse cecal contents samples. Methods MHV, Reo-3 and MNV were mixed with mouse cecal contents and used as reference samples,respectively. They were placed in the lysis buffer of RNA extraction reagent(buffer AVL)or normal saline, and stored at 4℃ and room temperature(22℃-25℃). RNA of these samples was extracted at 1,2,3,7,and 14 days. Then the amount of nucleic acid in samples was detected by real-time fluorescence quantitative PCR. Results A greater decrease of the amount of nucleic acid was observed when the samples were placed in normal saline than that kept in buffer AVL. The amount of nucleic acid in samples stored at 4℃ was found to be higher than that stored at 25℃ room temperature. The amount of nucleic acid in the samples which were kept in buffer AVL at 4℃ for 3 days was higher than 50%,still detectable in the samples kept for 7 days,and undetectable at 14 days. Conclusions Mouse cecal content samples are preferably stored in the lysis buffer of RNA extraction reagent and transported at 4℃ for the detection of MHV, Reo-3, and MNV nucleic acid. It is better to complete the detection test within 3 days.

Validation of the silencing site of marmoset B2m gene at the cellular level / 中国比较医学杂志

Objective To screen and determine the effective silencing targets of β2-microglobulin(B2m)gene at the cellular level in marmoset.Methods By homology comparison of the b2m gene in human and the B2m gene in marmoset, choose homology small hairpin RNA(shRNA)sequences targeting marmoset B2m gene were designed, We choose homology small hairpin RNA(shRNA)sequences targeting designed B2m gene to make homology analysis, and insert into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into HEK293T cells induced by polyethylenimine(PEI).The suppression of B2m mRNA was detected by real-time PCR.Results Two gene-silencing sequences were screened that lied in 290~310 bp and 665~685 bp of the marmoset B2m mRNA, and have statistical significance in the silencing rate:(46.54±7.91)% (P < 0.05) and(83.22±4.37)%(P < 0.0001).Conclusions Two effective silencing target sequences are screened at cellular level, which can be further used in studies on gene silencing in marmoset.

Comparison of different detection methods of monkey B virus antibody / 中国比较医学杂志

Objective Monkey B virus(BV), also known as Cercopithecine herpesvirus 1,is an important zoonotic pathogen.According to the national standard, antibodies are detected using BV as an antigen.However, the preparation of BV antigen is very stricted due to biosafety issues.Therefore, in this study, we used alternative antigens to detect the BV antibody by serological assay and verified their specifity and sensitivity.Methods A total of 135 blood samples from rhesus monkeys were tested by two ELISA method (BV and HVP2) and enzyme immunosorbent assay (EIA)method.The positive and suspicious samples were verified by immuno-fluorescence assay (IFA), Western blot and immunoblotting technique using HSV-1 gC1 purified glycoprotein as an antigen.Results The positive rates of HVP2-ELISA, BV-ELISA and HSV-1-EIA were 32.6%, 37.8% and 34.8%, respectively.Consistant result of the three detection method accounted for 91.1% (123/135), and the positive result were confirmed by IFA And WB.There were 12 suspicious samples,in which 33.3% (4/12) were verified to be positive.Conclusions Compared with BV antigen, the sensitivity and specificity of the alternative antigen HSV-1 are moe close than HVP2.Positive and suspicious samples should be verified by several method to avoid missed detection.

A current consideration of the microbes listed in the National Standard of Laboratory Animals for mice and rats / 中国比较医学杂志

In this paper, some items about the microbes listed in the current National Standard of Laboratory Animals were reviewed, including their host spectrum, impact of infection on the animals, their interference on research works and their epidemiology in laboratory animals.This paper may provide some clues for the update of our National Standard of Laboratory Animals.

Adeno-associated virus mediated p53 gene silence in marmosets / 中国比较医学杂志

Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique.Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis.The plasmids were transfected into African green monkey kidney cos-7 cells.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt.The adeno-associated virus-8 was injected through the hind leg vein.The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry.Results We screened two RNA interference effective arget sequences.The expression of p53 mRNA was suppressed ( 82.7 ±8.1 )% and ( 80.7 ± 7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3 ±11.5)% and (73.7 ± 10.7)%, respectively (P<0.05).The two marmosets after virus infection showed that there were virus distributions in the liver, testes, and neck detected by in vivo fluorescence imaging.The expression of p53 in the marmoset liver was detected by western blot, immunohistochemistry analysis showing no obvious changes.Conclusions In the present study, the decrease of P53 gene expression at cellular level is achieved, however, the liver P53 protein in the marmoset liver is not significantly changes.Further optimization of the way of infection is needed in the future.

Development of an amplified luminescent proximity homogeneous assay for detecting Sendai virus / 中国比较医学杂志

Objective To establish the amplified luminescent proximity homogeneous assay(AlphaLISA) for the detection of Sendai virus.Methods The antigen concentration,serum concentration and the donor beads/acceptor beads ratio used in the AlphaLISA method were optimumized by the phalanx experiments, then the antibodies of Sendai Virus in 40 rat sera were detected by the established AlphaLISA method and ELISA detection method.The results were compared and the differentia between the two methods was confirmed by IFA.Results The optimum concentration of SV bio-peptide in AlphaLISA assay was 250 nmol/L, the best proportion of donor beads/acceptor beads ratio was 1 ∶1, using the concentration of 20 μg/mL and the serum dilution was 1∶10000.7 of the 40 rat sera were detected SV positive by ELISA, the positive rate was 17.5%, 8 of the 40 rat sera were determined SV positive by AlphaLISA, and the positive rate was 20.0%, the AlphaLISA positive serum was confirmed by IFA.Conclusions We preliminary established the Amplified Luminescent Proximity Homogeneous Assay(AlphaLISA) for the detection of Sendai virus.The sensitivity of the method is comparable to classical ELISA method, but this method use less serum samples and without washing steps.The method has some advantages in degeneracy and accuracy.

Rhesus monkey P53 gene silencing at the cellular level / 中国比较医学杂志

Objective In order to establish a rhesus monkey model of p53 gene silencing, firstly we screened and determined the effective silencing targets of p53 gene at the cellular level in rhesus monkey.Methods The expression of p53 gene was detected in COS-7 cells ( derived from the kidney of the African Green Monkey, Cercopithecus aethiops).Three small hairpin RNA ( shRNA) sequences targeting rhesus monkey p53 gene were designed, analysed by bioinformatics, and inserted into lentivirus-based gene silencing constructs FUGW-TDT.The plasmids of p53-RNAi and control vector were transfected into the COS-7 cells, respectively.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western blot assay.Results p53 gene expression was detected in COS-7 cells.Bioinformatics analysis showed that three gene-silencing sequences were screened which lied in the open reading frame ( ORF) region and targeted 238 -258bp, 681 -701bp, 169 -189bp of the rhesus monkey p53 mRNA.At 48 hrs after transfection of the three silencing constructs, p53 mRNA was suppressed by(87.17 ±4.03)%, ( 72.62 ±4.11)% and(76.22 ±0.98 )%, and p53 protein was suppressed by ( 84.44 ±2.18 )%, ( 71.04 ±1.18)% and ( 74.17 ±0.95 )%, respectively. Conclusions We obtained three effective target sequences showing high efficiency in p53silencing, which can be used in further studies on gene silencing in rhesus monkey.

Establishment of an indirect immunofluorescence assay for detection of murine norovirus / 中国比较医学杂志

Objective To establish an indirect immunofluorescence assay for detection of murine norovirus ( MNV) .Methods Mouse leukaemic monocyte macrophage cell line RAW 264.7 cells were infected with MNV-1 and cultured for 36 hours to collect the virus and uninfected cells , and to make antigen glass slides .BALB/c mice were gavaged with MNV-1 (107 TCID50) and infected sera were collected as positive control .The serum was 1:10 diluted and used for measuring MNV antibody by immunofluorescence assay ( IFA ) .80 serum samples were tested using the two methods , IFA and ELISA, and the discrepant samples were validated by Western blotting .Results RAW264.7 cells were infected with MNV-1 for 36-48 h, showing an infection rate of 60% of the cells, and the cells infected for 36 h were preferred.IFA method was used to detect the serum with MNV-1 infection and showed that the antibody content was gradually increased at one week after infection , reaching a maximum antibody concentration at 4 weeks after infection , and maintained a stable level later .The mouse serum at four weeks after MNV-1infection was used as positive quality control . Among the 80 serum samples , 27 positive and 53 negative cases were detected by IFA method , and 32 positive and 48 negative cases were detected by ELISA .The five discrepant samples were verified by Western blotting , resulted in 3 positive and 2 negative cases . The coincidence rate of IFA was 96.0% and that of ELISA methods was 97.5%. Conclusions Basically, immunofluorescence assay can be used to detect the MNV-1 infection in mice, although false negative result may occur occasionally .IFA and ELISA detection can be selected as initial screening measures , and use Western blot assay to verify the discrepant samples .