Li Yijing

High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.

Expression of infectious pancreatic necrosis virus (IPNV) VP2–VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

Laboratory Observations Regarding Different Instars of Cyclosainsulana （Costa, 1834） （Araneidae） During Developmental Stages

Abstract The current experiment was conducted to find out the optimal conditions for mass rearing and developmental changes of Cyclosainsulana. The lab. conditions were maintained at (27±2)°C and (65±5)% RH. The clear perplex cages and natural diet consisting of the aphids, larvae of the house fly and larvae of drosophila were used for rearing. C. insulana took (123.12±7.26) days to develop from eggs to adults passing through eight instars under prevailing vivo conditions. The eggs were greenish white in color with average size of 0.57 mm ±0.034. The eggs spent (7.52±1.64) days in emergence. Maximum number of spiderlings survived at the 5th instar (84%) and minimum at the 1st instar (34%). The measurements of different body parts including the total body length, cephalothorax and pedipalps of the both male and female C. insulana were done with the help of micrometer and presented as mean± SD. The measurements varied in the each developing instar. It was concluded that spiders were difficult to rear in the lab. conditions and each developing stage which was regarded as instars showed variations in size colors and body characteristics.

Preparation and preliminary application of monoclonal antibodies against VP2 COE protein of infectious pancreatic necrosis virus

The recombinant protein IPNV VP2 was used as immunogen after purification by Ni-NTA.The 8-week-old BALB/c mice were intraperitoneally immunized with the VP2 protein for three times,then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice.Two hybridoma cell lines against the VP2 protein were obtained by screening with the indirect ELISA and limiting dilution assay,which were identified to be IgG1 subtype and named 5G10,5F3.The numbers of chromosomes of the two hybridomas were in the range of 75 to 120.Their antibody titers of cells culture supernatant were 1∶ 105,1∶ 102 respectively.Their titers of ascites were 1∶ 108,1∶ 104 respectively.Western-blot analysis and indirect immunofluorescence showed that the two McAbs could react with IPNV specificity.The 2 McAbs had no reactive capability with IHNV,VHSV,SVCV and HRV by the indirect ELISA.Antibodies additivity assay demonstrated that 5G10 and 5F3 recognized the different epitopes of IPNV VP2 nucleoprotein.We detected the clinical suffering from IPN rainbow trout liver tissue material,and the results confirmed that the 2 McAbs can be used for follow-up testing.

鱼源植物乳杆菌表达IPNV VP2-VP3重组蛋白及其口服免疫程序

为比较鱼源乳酸菌表达系统口服疫苗在不同免疫程序下诱导鱼免疫应答水平的差异，确定鱼源乳酸菌表达系统口服免疫虹鳟幼鱼的免疫程序。本研究构建了重组表达IPNV VP2-VP3蛋白的鱼源植物乳杆菌L1212，将重组菌pPG612-VP2-VP3/L1212包裹颗粒饲料，口服免疫虹鳟幼鱼。免疫程序分为连续免疫组、免疫1 d后间隔1 d再免疫1 d组、连续免疫4 d后32 d加强免疫1次组和间隔免疫后32 d加强免疫1次组。免疫后进行尾动脉采血，间接ELISA方法检测各组血清抗体效价，免疫接种66 d后，腹腔注射IPNV，计算各组相对免疫保护率。确定颗粒饲料按照1 mL/g 比例与108 CFU/mL 重组菌pPG612-VP2-VP3/L1212和2.5%海藻酸钠混合，于20 °C烘干制备口服疫苗。间隔免疫组血清抗体效价和攻毒保护率均显著高于其他组，并且免疫2次的间隔免疫组的血清抗体效价和攻毒保护率均显著高于免疫1次间隔免疫组。采用重组菌包被颗粒饲料饲喂虹鳟幼鱼，间隔免疫的免疫程序和免疫保护率优于连续免疫的免疫程序，对IPNV的入侵起到保护作用。

Abundance and Fluctuation in Spider Diversity in Citrus Fruits from Located in Vicinity of Faisalabad Pakistan

Abstract Spiders for the present study were collected from different fruit gardens (i.e. citrus) located at various localities (i.e., Tehsil Samundri, Jaranwala, Tandlianwala and Faisalabad) of District Faisalabad, Pakistan. Spiders belonging to six families and 33 species were captured from the two fruit gardens during the one year of this study. The citrus fruits garden was found to be best populated habitat as compared to other fruit garden. These sites were sampled by using pitfall traps; each month for five consecutive days from September 2010 to March 2011. As a result, 1 054 specimens were captured representing six families viz: lycosidae, thomosidae, gnaphosidae, saltisidae, araneidae and clubionidae. Lycosidae was more abundant, while clubionidae was less diverse during the study. Maximum population fluctuation among the spider specimens showed during the months from September and October, while the least abundance of spider specimens was reordered during June, November and December. Maximum taxonomic diversity was recorded from September to November, with the peak in September. It was concluded during these three months, when the citrus and guava gardens were attacked by the most of the pest insects. During the months of July and November diversity was moderate and mutually comparable, while in June and December, it was the least. This study contributed to the identification of spider diversity in the agro-ecosystem which could be used in the biological pest control.

Cloning of Porcine Lactoferrin Gene and Construction of Expression System in Recombinant Lactobacillus

Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin （PLFN）. A pair of primers was designed with Oligo6.0 and used to amplify PLFN gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR; the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLFN was created and identified by using plasmid extraction, PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393, Lactobacillus plantarum KLDS 1.0344, Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS 1.0413 by electroporation, and produced the recombinant strains of pPG612-PLFN/L, casei, pPG612-PLFN/L, plantarum, pPG612-PLFN/ L. paracasei and pPG612-PLFN/L, pentosus, respectively. The results indicated that PLFN gene had inserted into the expression vectors and achieved multiple Laetobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobaeillus

Prokaryotic expression of VP3 gene of infectious pancreas necrosis virus and antigenicity of expressed product

Infectious pancreatic necrosis(IPN) virus,the etiologic agent of infectious pancreatic necrosis in salmonid fish,causes significant losses to the aquaculture industry.The gene for the viral inner capsid protein(VP3) was amplified by RT-PCR method from IPNV,and cloned into pET30b vector.The expression of recombinant plasmid pET30b-VP3 in E.coli BL21(DE3) was induced and detected by SDS-PAGE analysis.The predicted molecular weight for unmodified r-trunc VP3 was approximately 30 ku and this was found to be the case for E.coli protein.The amount of expression made up 30 percent of the bacteria protein total expression by thin layer scanning analysis.The results showed that the VP3 gene of IPNV can express successfully in E.coli BL21.The fusion protein was purified with ProBondTM resin from the suspension centrifuged and the antisera against VP3 protein was produced.The pET30b-VP3 fusion protein can be recognized by the positive serum of IPNV by Western-blotting analysis.The prepared antisera reacted specifically with IPNV antigen by indirect ELISA.The antisera against VP3 protein had OD values at least twice that obtained for the negative control serum at a dilution of 1 :25 600.The results showed that the expressed VP3 protein was immunogenical and antigenical which is the same as the natural IPNV VP3 protein.In this experiment the IPNV VP3 protein was expressed successfully by using prokaryotic expression system.The expressed fusion protein was active and the antisera against VP3 protein were produced.