Analysis of drug - resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province in 2015 and 2016 / 中国血吸虫病防治杂志

ObjectiveTo investigate the drug-resistant gene polymorphisms in Plasmodium falciparum imported from Equatorial Guinea to Shandong Province. MethodsFrom 2015 to 2016, blood samples were collected from imported P. falciparum malaria patients returning from Equatorial Guinea to Shandong Province, and genome DNA of the malaria parasite was extracted. The drug-resistant Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum were amplified using a PCR assay, followed by DNA sequencing, and the sequences were aligned. Results The target fragments of all 5 drug-resistant genes of P. falciparum were successfully amplified and sequenced. There were 72.8%, 18.6%, and 8.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfcrt gene, respectively, and all mutant haplotypes were CVIET (the underline indicates the mutation site). There were 20.0%, 61.4% and 18.6% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfmdr1 gene, respectively, and the mutant haplotypes mainly included YF and NF (the underlines indicate the mutation sites). There were 1.4%, 98.6%, and 0 of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhfr gene, respectively, and AIRNI was the predominant mutant haplotype (the underline indicates the mutation site). There were 1.4%, 94.3%, and 4.3% of P. falciparum parasites carrying the wild-, mutant-, and mixed-type Pfdhps gene, respectively, and SGKAA was the predominant mutant haplotype (the underline indicates the mutation site). The complete drug-resistant IRNGE genotype consisted of 8.6% of the Pfdhfr and Pfdhps genes, and the K13 gene A578S mutation occurred in 1.4% of the parasite samples. Conclusions There are mutations in the Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and K13 genes of P. falciparum imported from Equatorial Guinea to Shandong Province, with a low frequency in the Pfcrt gene mutation and a high frequency in the Pfmdr1, Pfdhfr, and Pfdhps gene mutations, and the K13 gene A578S mutation is detected in the parasite samples.

[Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization].

OBJECTIVE: To prepare and purify the rabbit anti-TgMIC16 polyclonal antibody, so as to apply it in subcellular localization. METHODS: New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund's adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescence method. RESULTS: Indirect ELISA showed that the antibody titer was 1∶512 000. Western blotting showed that the recombinant TgMIC16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii. CONCLUSIONS: The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.

[Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2].

OBJECTIVE: To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression, and pay a way for Toxoplasma gondii nucleic acid vaccine development. METHODS: According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites, the HBsAg gene was amplified by PCR. The HBsAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene. The HBsAg-ROP2 fragment was amplified by PCR and digested with Hind Ⅲ and Kpn Ⅰ to clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2. The expression vector was transfected into HEK293T cells based on the identification of PCR amplification, restriction endonucleases and sequencing. RESULTS: The PCR product of HBsAg was about 700 bp, which was consistent with the theoretical value. Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3HBsAg-ROP2 recombinant plasmid. The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment. The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank. The target plasmid was successfully transfected into HEK293T cells, and the expression was correct, the protein concentration was 3.08 mg/ml. CONCLUSIONS: The recombinant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

Construction and expression of multi-gene recombinant plasmid pEG-FP-N1-HBsAg-ROP2 / 中国血吸虫病防治杂志

Objective To construct pEGFP-N1-HBsAg-ROP2 recombinant expression plasmid and transfect HEK293T cells for expression,and pay a way for Toxoplasma gondii nucleic acid vaccine development. Methods According to the HBsAg gene sequence and pcDNA3-p30-ROP2 recombinant plasmid restriction sites,the HBsAg gene was amplified by PCR.The HB-sAg gene was cloned into the pcDNA3-p30-ROP2 and instead of p30 gene.The HBsAg-ROP2 fragment was amplified by PCR and digested with HindⅢand KpnⅠto clone into the pEGFP-N1 eukaryotic expression vector and construct the recombinant pEGFP-N1-HBsAg-ROP2.The expression vector was transfected into HEK293T cells based on the identification of PCR amplifi-cation,restriction endonucleases and sequencing.Results The PCR product of HBsAg was about 700 bp,which was consis-tent with the theoretical value.Two bands of about 5.4 kb and 1.9 kb were obtained after double enzyme digestion with pcDNA3-HBsAg-ROP2 recombinant plasmid.The recombinant plasmid pEGFP-N1-HBsAg-ROP2 was double-digested to generate an empty vector fragment of about 4.7 kb and a band of about 1.9 kb of HBsAg-ROP2 fragment.The results of sequencing showed that the sequence was 99.84% identical with the published sequence in GenBank.The target plasmid was successfully transfect-ed into HEK293T cells,and the expression was correct,the protein concentration was 3.08 mg/ml.Conclusion The recombi-nant plasmid pEGFP-N1-HBsAg-ROP2 is successfully constructed and expressed efficiently.

Preparation and purification of polyclonal antibody against Toxoplasma gondii microneme protein 16 and its application in subcellular localization / 中国血吸虫病防治杂志

To prepare and purify the rabbit anti-TgMIC16 polyclonal antibody, so as to apply it in subcellular localization.New Zealand white rabbits were immunized with purified recombinant TgMIC16 mixing with the same volume of Freund’s adjuvant for three times, respectively. The rabbit serum was collected on the 14th day after the last immunization. The polyclonal antibody in rabbit serum was purified with Protein A affinity purification column. ELISA and Western blotting were used to detect the antibody titer and specificity of polyclonal antibody. The polyclonal antibody was used to the localization of TgMIC16 by the immunofluorescence method.Indirect ELISA showed that the antibody titer was 1∶512 000. Western blotting showed that the recombinant TgMIC16 protein was recognized by the specific polyclonal antibody. IFA showed that TgMIC16 was located in the microneme of Toxoplasma gondii.The rabbit anti-TgMIC16 is prepared and purified, and successfully applied to immunofluorescence localization of TgMIC16 in T. gondii.

[Allele genetypes and homology analysis of MSP-1 and CSP gene of Plasmodium vivax in Shandong Province].

OBJECTIVE: To analyze the genotypes and homology of MSP-1 and CSP gene of Plasmodium vivax in Shandong Province, so as to provide the evidence for case traceability. METHODS: A total of 12 blood samples were collected from P. vivax-infected cases in Shandong Province in 2011. Parasite genomic DNA was extracted. Primers were designed according to MSP-1 and CSP gene sequences of P. vivax. Then Nested PCR, enzyme digestion, sequencing and sequence alignment, and homologous analysis were performed. RESULTS: The MSP-1 gene of all the 12 samples from P. vivax-infected cases were detected with a 470 bp PCR amplification band, and 350 bp and 120 bp enzyme digestion fragments, which were identified as type Sal-1. An analysis of phylogenetic tree of MSP-1 gene showed that the sequences of 9 indigenous case samples in Shandong Province were located in the same branch, one case sample infected from India was located in the same branch with India strains. All the 12 P. vivax-infected samples covered GDRA (D/A) GQPA sequences in CSP gene, which were identified as type PV-Ⅰ. Of the CSP gene among 12 P. vivax-infected samples, 10 samples of indigenous case in Shandong Province and one sample of the case infected in Guangdong Province were detected with both 560-840 bp and 150-230 bp PCR amplification bands, which were identified as temperate zone family strain of type PV-Ⅰ. However, one sample from the case infected in India was detected only with a 560-840 bp band, which was identified as tropical zone family strain of PV-Ⅰ. An analysis of phylogenetic tree of CSP gene showed that the sequences of 10 samples from the indigenous cases in Shandong Province and one sample from the case infected in Guangdong Province were located in the same branch, one sample from the case infected in India was located in the same branch with India and Indonesia strains. CONCLUSIONS: Of all the indigenous isolates in Shandong Province, MSP-1 gene is genotyped type Sal-1, CSP gene is genotyped temperate zone family strain of type PV-Ⅰ, with a high homology found among the indigenous isolates.

Characteristics of Imported Malaria and Species of Plasmodium Involved in Shandong Province, China (2012-2014)

Malaria remains a serious public health problem in Shandong Province, China; therefore, it is important to explore the characteristics of the current malaria prevalence situation in the province. In this study, data of malaria cases reported in Shandong during 2012-2014 were analyzed, and Plasmodium species were confirmed by smear microscopy and nested-PCR. A total of 374 malaria cases were reported, 80.8% of which were reported from 6 prefectures. Of all cases, P. falciparum was dominant (81.3%), followed by P. vivax (11.8%); P. ovale and P. malariae together accounted for 6.4% of cases. Notably, for the first time since 2012, no indigenous case had been reported in Shandong Province, a situation that continued through 2014. Total 95.2% of cases were imported from Africa. The ratio of male/female was 92.5:1, and 96.8% of cases occurred in people 20-54 years of age. Farmers or laborers represented 77.5% of cases. No significant trends of monthly pattern were found in the reported cases. All patients were in good condition after treatment, except for 3 who died. These results indicate that imported malaria has increased significantly since 2012 in Shandong Province, especially for P. falciparum, and there is an emergence of species diversity.