Preliminary study on rapid identification the species of Mycobacteria by PCR-restriction fragment length polymorphism based upon hsp65 gene / 中华流行病学杂志

Objective To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level. Methods hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae Ⅲ and Bstp Ⅰ, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed. Results A total of 40 Mycobacterium species (strains) was analyzed, in which six reference strains of Mycobacterium tuberculosis Complex had two different electrophoresis patterns, and thirty-four reference species of non-tuberculosis Mycobacteria had unique pattern. Conclusion PCR-RFLP Based upon hsp65 gene can be used for identification of Mycobacterium species, and the method is more rapid and simple and easy-to-use for mycobacterial species identification.

Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice.</p><p><b>METHODS</b>Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens.</p><p><b>RESULTS</b>The purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group.</p><p><b>CONCLUSIONS</b>Ag85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.</p>

Effect of Mycobacterium smegmatis vaccine on the level of nitric oxide produced by peritoneal macrophages in mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice.</p><p><b>METHODS</b>Balb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro. The supernatants were collected and the concentrations of NO were analyzed through the reaction with Griess reagents.</p><p><b>RESULTS</b>The levels of NO produced by the peritoneal macrophages in the control group, low-dose group, middle-dose group, and high-dose group were (3.50 +/- 3.11), (16.63 +/- 6.47), (13.97 +/- 6.20), and (7.55 +/- 2.26) ng/ml, respectively. The levels of NO in all dosing groups were significantly different from that in control group (P < 0.01).</p><p><b>CONCLUSION</b>Mycobacterium smegmatis vaccine can promote the peritoneal macrophages to produce NO in mice.</p>

Study on mutation of presenilin-1 gene in familial Alzheimer's disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the role of the mutation of presenilin-1 exon 6 in pathogenesis of Alzheimer's disease(AD) patients.</p><p><b>METHODS</b>Exon 6 of presenilin-1 was analyzed by use of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA analyzer technique in 2 patients with familial AD, 53 patients with sporadic DA, 60 patients with vascular dementia(VD) and 90 normal controls.</p><p><b>RESULTS</b>Mobility shift of SSCP in exon 6 of presenilin-1 was detected in 2 cases with FAD, 4 cases with SDA and 1 case with VD. Two missense mutations were found in the patients by DNA sequence analysis, one mutation was 1123 nt C-->G(Cys 23 Trp) and the other was 1300 nt A-->C(Asp 200 Ala).</p><p><b>CONCLUSION</b>Mutations in exon 6 of presenilin-1 existed in the patients with FAD and SDA, and the two missense mutations were probably pathological by nature.</p>