OBJECTIVETo develop an allele-specific PCR (AS-PCR)/restriction fragment length polymorphism (RFLP) assay for CYP1B1 gene haplotypes predisposing to primary congenital glaucoma (PCG).

METHODSTwenty Chinese PCG patients and 20 healthy controls were recruited. Peripheral blood sample was subjected to direct sequencing for common single nucleotide polymorphisms (SNPs) of the CYP1B1 gene. Based on the results, CYP1B1 gene haplotypes were constructed by PCR-RFLP and AS-PCR combined with RFLP.

RESULTSFour SNPs loci were identified by sequencing, which included rs10012 G>C (S1 in exon 2), rs1056827 T/G (S2 in exon 2), rs1056836 C/G (S3 in exon 3) and rs1056837T>C (S4 in exon 3). The distribution of such loci showed different characteristics between the two groups. 50% of the PCG patients had rs10012 G>C and rs1056827 T>G, while 25% of PCG patients had rs1056836 C>G and rs1056837T>C. As for the controls, 25% had rs10012 G>C and rs1056827 T>G, 10% had rs1056836 C>G and rs1056837T>C. None of the SNP loci has presented alone. PCR-RFLP was carried out to confirm the results of SNPs typing, but could not confirm the linkage between the SNP loci. By contrast, AS-PCR combined with RFLP has achieved specific amplification for rs10012 G>C and thorough differentiation of 1056827 T>G polymorphism. Similar results have been obtained by the same method for rs1056836 C>G and rs1056837T>C typing and linkage disequilibrium analysis.

CONCLUSIONThe AS-PCR/RFLP assay has successfully constructed the haplotypes of the CYP1B1 gene. For its accuracy, efficiency and specificity, the method may be used for constructing haplotypes for hereditary disease studies.

Adult ; Aged ; Alleles ; Base Sequence ; Cytochrome P-450 CYP1B1 ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; genetics ; Genotype ; Glaucoma ; congenital ; genetics ; Haplotypes ; Humans ; Linkage Disequilibrium ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Reproducibility of Results ; Sequence Analysis, DNA ; methods

Objective To evaluate the immunogenicity and safety of inactivated poliovirus vaccine derived from Sabin strain (sIPV) in Banna mini pigs, and to provide experimental evidence for the new animal model. Methods sIPV vaccines which are listed at Institute of Medical Biology at Chinese Academy of Medical Sciences were used in this study. The groups of intramuscular sIPV and the wild strain IPV injections (IPV derived from wild strain, wIPV) were designed, and the saline group was used as a negative control group. The Banna mini pigs in various groups were immunized at 0, 1 and 2 months. Blood samples were collected before immunization and on days 30 after each immunization. Levels of neutralizing antibodies were tested for evaluating immunogenicity. The safety was evaluated by observation of the status and weight of mini pigs. Results After the three dose immunization schedules in the Banna mini pigs, the seroconversion rates of type Ⅰ,Ⅱ and Ⅲ sIPV experimental groups and wIPV group were all up to 100%. The neutralizing antibody levels in all the three types were much higher than the protective titer 1: 8. The weight of mini pigs increased after vaccination. Conclusions The sIPV vaccine has good immunogenicity and safety in Banna mini pigs. Banna mini pigs could be a new animal model for evaluation of sIPV vaccine.