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Construction of the prokaryotic expression system for the prtC gene of Porphyromonas gingivalis and analysis of the relationship between thelocal level of PrtC protein and the periodontal damage

Ping RUAN ; Yaodong LUO ; Jie YAN

Chinese Journal of Zoonoses.2005;(5):369-374. doi:10.3969/j.issn.1002-2694.2005.05.001

Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.

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Morphological characteristics of eggs of Trichuris in humane, domestic and wild animals

Bingzhen FAN

Chinese Journal of Zoonoses.2005;(7):545-547. doi:10.3969/j.issn.1002-2694.2005.07.001

The whipworm,Trichuris trichiura,has a worldwide distribution but is more common in tropical and subtropical areas and in regions where sanitation is poor.The common name of whipworm is most descriptive,the thick posterior part of the body forming the stock,and the long thin anterior portion.The adult worma are from 3 to 5 cm long,the female worm is longer than the male worm.On the posterior part is pinkish gray and contains the intestine and reproductive organs. Diagnosis is by demonstraion of the characteristic barrel-shaped eggs (50-54 by 22-23μm)usually found in the stool.But their morphology of adult worms and eggs among the human,domestic and wild animals are quite different to diagnosis.The females are larger(35-55 mm long)than the males(30-45 mm).Each female worm can produce 3,000 to 70,000 eggs daily.The eggs are lemon-like with a plug at each end.Their size are 50-54 μm and 22-23 μm respetively.

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Immunoprotection of recombinant antigen LTB-UreB-HpaA in Helicobacter pylori infected mice and immunoenhancement of the inner adjuvant LTB

Dongjiao LUO ; Zhexin SHAO ; Shuilin XU ; Jie YAN

Chinese Journal of Zoonoses.2005;(8):645-651. doi:10.3969/j.issn.1002-2694.2005.08.002

In order to increase antigenicity of H. pylori-specific antigens and decrease the cost of further industrial production, we used a special PCR with linking primers to construct a fusion gene containing H.pylori ureB and hpaA genes and E.coli ltB gene, and to costract its prokaryotic expression system pET42a-ltB-ureB-hpaA-E.coliBL21DE3. The sequencing result indicated the 100% nucleotide sequence homology of the constructed ltB-ureB-hpaA fusion gene compared to those of the original separated genes. Output of the target recombinant protein rLTB-UreB-HpaA was approximate 15% of the total bacterial proteins measured by SDS-PAGE. The rLTB-UreB-HpaA could induce the immunized rabbits to produce specific antibodies with immunodiffusion titer of 1∶8, and could combine to the commercial rabbit antibody against the whole cell of H.pylori as well as rabbit anti-UreB and anti-HpaA sera by using Western bolt assays. Using GM1-ELISA, the ability of rLTB-UreB-HpaA binding to bovine GM1 was confirmed.And rLTB-UreB-HpaA (200 μg per mouse) could prevent 100% of the immunized BaLb/C mice from H.pylori strain SS1 infection. The co-administration with 10 μg rLTB, the rUreB or rHpa could increase its protective rates in the immunized mice from 66.7% to 81.8% and 83.3%, respectively. All these data leads a conclusion that rLTB-UreB-HpaA is a great potential as a practical genetic engineering vaccine to prevent H.pylori infection.

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Highly sensitive Immuno-PCR assay for the detection of the circulating antigens in trichinellosis

Hui LI ; Bianli XU ; Xudong ZHAO ; Yan DENG ; Ximeng LIN

Chinese Journal of Zoonoses.2005;(9):769-775. doi:10.3969/j.issn.1002-2694.2005.09.010

A highly sensitive immuno-PCR assay based on sandwich ELISA and PCR was developed to detect the circulating antigen in trichinellosis. Antigens were purified from the muscle larvae of Trichinella spiralis, and the myeloma cells were fused with spenocytes immunized with T. spiralis antigens to product the specific monoclonal antibodies. Indirect ELISA was used to select the antibody-secreting hybrodoma cells. By this method of procedure, monoclonal antibody F4C6 against the T. spiralis ES antigen was obtained, which was used as the indicator antibody, while the rabbit polyclonal antibodies against T. spiralis were to be used as capturing antibodies. The plasmid Bluecript Ⅱ KS was amplified by PCR with biotin-labeled primer M13-20, and thus the biotin-labeled DNA was obtained. Both the second antibody and DNA labeled with biotin were to be linked with 100 ng/ml avidin. The whole procedures of assay consisted of two steps, in which the circulating antigens were captured by monoclonal antibody through sandwich ELISA in the first step, and the DNA linked by monoclonal antibody was amplified by PCR in the second step. The sensitivity of this method was compared with that of the ELISA assay. It was found that the measuring ranges to detect the circulating antigens in trichinellosis were 50 pg/L to 0.005 pg/L for the immuno-PCR assay, and 5 μg/L to 0.05 μg/L for ELISA assay, the former was quite higher than that of the latter. It is evident that this method is highly sensitive for the detection of circulating antigens in trichinellosis.

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Over-expression and purification of the recombinant p30 antigen of Toxoplasma gondii

Dianbo ZHANG ; Defu ZAI ; Maoqing GONG ; Jin LI ; Qingkuan WEI ; Yong CUI ; Bingcheng HUANG ; Keyi LIU

Chinese Journal of Zoonoses.2005;(12):1089-1093. doi:10.3969/j.issn.1002-2694.2005.12.015

To provide the basis for preparation of diagnostic kits and vaccines in Toxoplasma gondii infection, the gene coding for the qualified recombinant p30 protein (SAG1) of this parasite was amplified by PCR, and the amplified gene was cloned into prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid, and then transformed to E.coli DH5α. The positive recombinant plasmid was screened by PCR and double enzymes digestion, and the nucleotide sequence of p30 gene was determined by automated DNA sequencing. Meanwhile, the identified recombinant plasmid was transformed to E.coli BL21(DE3) with the expression of p30 on bacteria induced by IPTG and the expressed protein was identified by SDS-PAGE. The protein obtained was then further purified and refolded, and its biological activity was checked by Western blotting. It was shown that the size of the amplified gene was 750 bp with molecular weight of 30 ku, and this protein could specifically react with monoclonal antibody against p30 protein.

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High level expression and purification of 1 C-terminal fragment of merozoite surface protein of Plasmodium falciparum in Pichia pastoris

Zhongguang ZHANG ; Hengmei ZHAO ; Yuxiang GONG

Chinese Journal of Zoonoses.2005;(12):1047-1051. doi:10.3969/j.issn.1002-2694.2005.12.005

To obtain an ideal recombinant C-terminal fragment of the merozoite surface protein of Plasmodium falciparum in the Pichia pastoris expression system, the major surface protein-119 (MSP-119) gene sequence bearing the 6-his gene was inserted into expression vector pPIC9k and the target gene was transformed to the susceptible yeast cells GS115 by using electroporation. The multiple inserts were screened and the successfully expressed MSP-119 protein with the relative molecular weight of 12kDa in the supernatants of cell cultures could be detected by SDS-PAGE. Meanwhile, Western blot analysis also demonstrated that this protein reacted with mouse anti-MSP-119 monoclonal antibody, and the expression level of MSP-119 was more than 1.0 g/L. It is concluded that this recombinant protein expressed in the Pichia pastoris expression system resembles the native proteins existed.

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Cysticercosis in Domestic Animals and Rodents

Fan P.C. ; Chung W.C. ; Guo J.X. ; Ma X.Y. ; Wang I.C.

Chinese Journal of Zoonoses.2006;(1):1-10. doi:10.3969/j.issn.1002-2694.2006.01.001

Cysticercus cellulosae, C. bovis, and C. taiwanensis are the three species of cysticercosis of human taeniid with their adults worms Taenia solium, T. saginata saginata and T. s. asiatica respectively. C. cellulosae is prevalent in America, Europe and Asia, C. bovis in Africa, America and Asia and C. taiwanensis in Asia. The natural infection source is pig for C. cellulosae, cattle for C. bovis and pig and wild boar for C. taiwanensis. The predilection sites are muscles for C. cellulosae and C. bovis and liver for C. taiwanensis. While the prepatent periods of these three species were 60-70 days, 60-75 days and 28 days respectively. Most C. cellulosae and a few for C. bovis and C. taiwanensis survive in pig, while most C. bovis and a few for C. taiwanensis survive in cattle. In rodent, all three species have a long life. C. cellulosae and C. bovis migrate through blood and lymph vessels to the small intestinal wall and then to the body muscles while C. taiwanensis migrate through blood vessels to the small intestinal wall and then to the liver. The minimal effective dosage of praziquantel against cysticercosis should be 100 mg/kg daily for three consecutive days.

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Reconstruction of the heat-labile enterotoxin subunit B gene of Escherichia coli and its immune adjuvant activity on mucosa

Juyun SUN ; Panli TAN ; Ye HU ; Yafei MAO

Chinese Journal of Zoonoses.2006;(5):385-390. doi:10.3969/j.issn.1002-2694.2006.05.001

To reconstruct the heat-labile enterotoxin subunit B (LTB) gene of Escherichia coil in order to increase the outputs of the prokaryotic expression on recombinant LTB (rLTB), and to determine its immune adjuvant activity on mucosa,the nucleotide sequence of the whole length of LTB gene was synthesized according to the preferred codons of E. coli, and the prokaryotic expression plasmid pET32a-rLTB and its expression system in E. coli BL21DE3 were reconstructed. The recombinant plasmid was extracted and the inserted sequence of rLTB gene was determined. Meanwhile, the expression quantity of the reconstructed rLTB was identified by SDS-PAGE and BioRad agarose image analyzing system, and compared with that of the un-reconstructed rLTB. The abilities of the reconstructed rLTB and the un-reconstructed rLTB to bind with bovine GM1 were determined by means of GM1-ELISA assay. By using the recombinant urease subunit B as antigen, the effects of the reconstructed and the un-reconstructed rLTB on the improvement of immune protection of BALB/c mice infected with Helicobacter pylori strain SS1 and the induction of S-IgAs in infected mice were assayed. The experimental results showed that the expression quantity of the reconstructed rLTB approached upto 35.4% of the total bacterial proteins after induction with 1 mmol/L IPTG for pET32a-rLTB-E. coliBL21DE3 and to be 12.6 times higher than that of the un-reconstructed rLTB (2.8 %). In addition, both the abilities of the recombinant reconstructed LTB and the un-reconstructed rLTB to bind with bovine GM1 could be demonstrated by GM1-ELISA. The immune protection rate of the recombinant urease subunit B in the infected mice was 66.7%; and it could reach up to 91.7% with a significantincrease of the specific S-IgA level, when it was immunized with the reconstructed or the un-reconstructed rLTB. It is concluded that the reconstructed LTB gene in the present study shows a remarkable increased outputs of expression of this gene with a strong immune adjuvant activity on mucosa.

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Expression and identification of the multiple gene ROP2-P30 of Toxoplasma gondii in E.coli BL21

Dianbo ZHANG ; Qingkuan WEI ; Defu ZAI ; Yong CUI ; Jin LI ; Honggang GAO ; Xuelian BAI ; Lijiang ZHAO ; Guangdong HAN ; Keyi LIU

Chinese Journal of Zoonoses.2006;(6):538-543. doi:10.3969/j.issn.1002-2694.2006.06.013

To obtain the functional fusion protein of rhoptry protein 2, compound rhoptry protein2 and surface antigen 1 of Toxoplasma gondii. the ROP2 and P30 genes from genomic DNA of T.gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector. Then the ROP2 fragment was subcloned to pET-30a(+) plasmid digested by EcoRⅠand Hind Ⅲ to construct plasmid pET-ROP2. Furthermore,the P30 fragment was subcloned into pET-ROP2 digested by BglⅡand EcoRⅠto create plasmid pET-ROP2-P30, the resulting recombinant plasmids , transformed into E.coli BL21 (DE3), were induced with IPTG. and the proteins identified by SDS-PAGE were further purified and refolded. The biological activity was analyzed by Western blot with specific antibody. It was found that the sizes of ROP2 and ROP2-P30 were 1212 and 1896bp with corresponding molecular weight 50- kDa and 75-kDa, respectively. The recombinant protein ROP2 (50-kDa) could specifically react with rabbit-polyclonal antiserum, and complex fusion protein ROP2-P30 (75- kDa) could react with P30 monoclonal antibody.

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Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli

Jianhua XU ; Jiayong ZHU ; Xiaobao JIN ; Qinying XU ; Leishan LIU ; Yan MA ; Yan WANG

Chinese Journal of Zoonoses.2007;(4):311-318. doi:10.3969/j.issn.1002-2694.2007.04.001

In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC,and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.

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