the 30k - family proteins are lipoproteins synthesized by the fat body in the silkworm and display a molecular weight of approximately 30kd [ 13 ] . they are highly expressed in silkworm larvae during the early stage of the fifth instar and during the pupation stage of development , where they are predominantly located in the hemolymph as a major source of energy for growth , development , and pupation . in addition , the 30k - family proteins found in the silkworm are highly conserved , share more than 41% amino acid sequence homology , and display similar tertiary structures with the common conserved domain of lipoprotein-11 . moreover , the 30k family of proteins was found to have an antifungal activity . found that the purified recombinant glutathione - fused 30k protein lp6g1 inhibited the morbidity of silkworm pupae and prolonged the life of the fungus - infected silkworm chrysalis . thus far , several 30k proteins were also reported to have an antiapoptotic activity [ 611 ] . park et al . demonstrated that the purified 30kc6 protein that was expressed in escherichia coli , enhanced cell viability , and inhibited apoptosis of both virus - infected sf9 cells and chemical - treated hela cells . discovered that the purified escherichia coli expressed 30kc19 protein enhanced cell viability and prevented cell apoptosis in actinomycin - induced sf9 cells . wang et al . showed that 30kc19 expressing chinese hamster ovary cells increased both the quantity and biological activity of human erythropoietin . previous studies by us have shown that the purified 30k proteins expressed in the insect system encoding 30kc6 , 30kc12p , and 30kc19g1 inhibited apoptosis in h2o2-induced human umbilical vein endothelial cells ( huvec ) .   in the current study , both slp and lsp - t were initially screened from a gene library of the silkworm by comparative homology , where it was found that both slp and lsp - t shared a high degree of amino acid sequence homology and a predicted tertiary structure consistent with the reported silkworm family of 30k proteins . furthermore , slp and lsp - t were cloned , expressed , and purified . in novel experiments , we studied the effects of slp and lsp - t on h2o2-induced apoptosis silkworm bmn cells and huvec cells by analyzing both the cell viability and mechanisms of cell death . the e. coli strain tg1 , the dh10bac strain , the pfastbac htb vector , and cultured silkworm bmn cells were conserved in our laboratory . a tanon-1600 gel image system was purchased from shanghai tianneng science and technology limited company ( shanghai , china ) . mouse 6  his - tag monoclonal antibody and the cell death detection elisa kit were purchased from roche life science , inc . horseradish peroxidase ( hrp)-conjuagated goat - antimouse - igg was purchased from beijing dingguo biotechnology company ( beijing , china ) . serum - free insect cell culture medium sf-900ii sfm , mammalian high glucose culture medium dmem , fetal bovine serum ( fbs ) , and hank 's balanced salts solution ( hbss ) buffer were all obtained from gibco / invitrogen ( usa ) . the liposomal transfection reagent , cellfection ii reagent , and the ni - nta purification system were also obtained from invitrogen ( usa ) . silkworm slp and lsp - t were screened , and their cdna sequences were obtained by the online available blast tool ( http://blast.ncbi.nlm.nih.gov/ ) . the cdna open reading frames ( orf ) of the targeted genes were obtained by the dnastar software program . dna sequences analysis of slp and lsp - t and homology comparisons with three reported 30k - family proteins , including 30kc6 [ 6 , 12 , 13 ] , 30kc12p , and 30kc19g1 [ 8 , 9 ] were carried out using the clustal method of megalign in dnastar . homology comparisons of the amino acid sequences present in slp and lsp - t were conducted by the single version clustx1.8 and bioedit software programs according to the protein sequences and functions of the three reported 30k - family proteins . swiss - model ( http://swissmodel.expasy.org/ ) and rasmol version2.7 were employed to predict the tertiary structures . pcr primers were designed according to the screened sequences of both slp and lsp - t as shown below : p1 : 5-cacggatccatgaagtttttggtgtttttc-3 ; p1 : 5-cccaagcttttagttgaggattgagatagac-3p2 : 5-atcggatccatgaagaccttggcgg-3p2 : 5-cgcaagcttttagttgaggatcgagatag-3. note that the bamh i and hind iii restriction enzyme digest sites were introduced and indicated by the underlined text .    5-cacggatccatgaagtttttggtgtttttc-3 ;    5-cccaagcttttagttgaggattgagatagac-3   5-atcggatccatgaagaccttggcgg-3   5-cgcaagcttttagttgaggatcgagatag-3. note that the bamh i and hind iii restriction enzyme digest sites were introduced and indicated by the underlined text . total rna was extracted from the fat body of a 5th instar silkworm larvae by grinding the collected tissues in liquid nitrogen . cdna was obtained using an rt - pcr kit and by using the p1/p1 and p2/p2 primers , respectively . both slp and lsp - t were amplified , purified and then cloned into t - easy vectors , respectively . the recombinant plasmids were identified by restriction enzyme analysis using bamh i and hind iii . the constructed t - easy vectors including sequences of both slp and lsp - t , and the transfer vector pfastbachtb , were digested with the same restriction enzymes described above and ligated . e. coli dh10bac competent cells were transformed with the constructed recombinant transfer vectors and subsequently cultured on lb media containing iptg , x - gal , gentamycin , kanamycin , and tetracycline . positive clones were identified by pcr using forward and reverse m13 primers : forward primer is 5-cccagtcacgacgttgtaaaacg-3. reverse primer is 5-agcgataacaatttcacacagg-3. the screened recombinant dna for bacmid - slp and bacmid - lsp - t was transfected into bmn cells by a liposome - mediated method and the virus titer determined by the tcid50 ( 50% tissue culture infective dose ) cytopathic effect endpoint dilution assay , which quantifies the amount of virus required to kill 50% of infected cultured host cells . bmn cells were infected with the harvested second generation viruses at a multiplicity of infection ( moi ) of 10 . the infected cells were harvested 72  h following viral infection and washed once with pbs . cells were resuspended in pbs and lysed by an ice - bath ultrasonic sound method . the lysates were centrifuged for 15  min at 12000  g under 4c using a beckman centrifuge ( a1330011 ) , and the supernatants were harvested and incubated with a ni - nta agarose affinity filler on a platform orbital shaker overnight . the collected samples were washed in 20  mm imidazole and 250  mm imidazole three times , respectively . the purification of proteins was conducted according to the manufacturer 's instructions ( invitrogen ) of the ni - nta kit . a mouse 6  his - tag monoclonal antibody was used to identify the target proteins by western immunoblotting . bmn cells were used in the logarithmic growth phase and plated in 96-well microtiter plates at a density of 1  10 cells / well and cultured overnight . appropriate dilutions of h2o2 were made in hbss buffer at 0.1  mm , 0.5  mm , and 1  mm , respectively . the cell culture media were discarded from the 96-well plates , which were washed once with hbss prior to adding the appropriate dilutions of h2o2 to the wells . h2o2-treated cells were cultured for 10  min , 30  min , or 60  min in an incubator set at a temperature of 27c . h2o2 was discarded , and the cells were washed twice with hbss . finally , cells were added to regular culture media , supplemented with fbs with a 10% final concentration for 24  h at 27c . bmn cells in the logarithmic phase of growth were plated in 96-well microtitre plates at a density of 1  10 cells / well and cultured overnight at 27c . cultured cells were pretreated with purified slp and lsp - t at a final concentration of 1.5  g / ml for 24  h. bmn cells were then treated with 0.5  mm h2o2 for 10  min to induce apoptosis , following which , cells were cultured in complete media containing slp and lsp - t for an additional 24  h. purified 30kc6 protein was used as a positive control . normal cultured bmn cells and h2o2-treated bmn cells served as control groups . the treated bmn cells were pulsed with 10  l of mtt reagent ( 5  mg / ml ) per well of a 96-well microtitre plate and cultured for 4  h in the incubator and in the dark at 27c . next , the culture media was discarded , and 150  l of dmso was added to each well to a final concentration of 0.1% . after shaking for 10  min , the absorbance of each well was measured at a wavelength of 490  nm to calculate the relative cell viability ratio . for quantitative determination , dna fragmentation was evaluated by histone - associated dna fragments using a photometric enzyme immunoassay ( cell death detection elisa , roche life sciences , inc . ) , according to the manufacturer 's instructions . the treated bmn cells were harvested , and the cell suspension was pelleted by centrifugation . after centrifugation , 10  l volumes of the supernatants were added to streptomycin - coated culture plates . each culture plate was then incubated with 80  l of specific antibody for 2  h. after washing three times in pbs , samples were measured by an elisa format procedure and spectrophotometry to read the absorbance values of each well . huvecs were propagated to the logarithmic growth phase , seeded into 96-well microtiter plates at a density of 1  10 cells / well , and cultured overnight at 37c with 5% co2 . the cells were washed once in hbss and stimulated with different concentration of h2o2 ( 0.25  mm , 0.5  mm , and 1.0  mm ) for 10  min , 30  min , or 60  min in an incubator set at a temperature of 37c . culture media containing h2o2 were discarded , and cells were washed twice in hbss buffer . finally , cells were cultured in normal culture media with 10% fbs in the incubator with 5% co2 for 24  h at 37c . huvec cells were pretreated with slp and lsp - t as described previously , following which , cells were treated with 0.5  mm h2o2 for 30  min . next , the cells were incubated with either slp or lsp - t at a final concentration of 4  g / ml and cultured for 24  h. control groups were set up exactly as described for the cultured bmn cells . cell viability in the treated huvec cells was conducted exactly as described previously and by the mtt reagent assay . dna fragmentation analysis in huvec cells was conducted as described previously by an elisa assay . data described in this paper are shown as mean  sd . statistical significance between means was determined and analyzed by one - way analysis of variance ( anova ) and student 's t - test using spss version 13.0 software . a p value < 0.05 was considered statistically significant , and a p value < 0.01 was considered highly significant . there are only several 30k - family proteins reported to be expressed in the silkworm . to identify new members of the 30k family of proteins , a gene library derived from the silkworm was screened with the clustalx 1.8 software program by comparative homology analysis against the reported 30k family of proteins ( figure 1 ) . this analysis revealed that both slp and lsp - t shared more than 41% homology with the already characterized 30k family of proteins ( figure 1(b ) ) . furthermore , there were conserved domains of lipoprotein-11 in both slp ( amino acids 6261 ) and lsp - t ( amino acids 6266 ) and the other reported 30k family of proteins . tertiary structures of both slp and lsp - t were further predicted by applying swiss - model software ( figure 2 ) , which showed that the predicted tertiary structures of slp and lsp - t were similar to that of the reported 30k family of proteins . these data indicate that both slp and lsp - t are novel candidates of the 30k family of proteins in the silkworm .   in order to identify the recombinant bacmid , we extracted dna of the recombinant bacmid and subjected it to pcr analysis using m13 primers and the targeted slp and lsp - t gene primers , respectively . the cross pcr results demonstrated that there were four pcr products with sizes of 3220  bp , 2620  bp , 1480  bp , and 789  bp in the analysis of bacmid - slp ( figure 3 ) . in addtion , there were four pcr products with sizes of 3230  bp , 2630  bp , 1490  bp , and 804  bp in the analysis of bacmid - lsp - t ( figure 3 ) . these data were consistent with the predicted results and thus indicated that both recombinant bacmid - slp and bacmid - lsp - t were correct and could be used in further functional studies .   in order to study the function of slp and lsp - t , recombinant bacmid - slp and bacmid - lsp - t next , the bmn cells that expressed the bacmid - slp and bacmid - lsp - t constructs were lysed , and the supernatants were collected and purified with the ni - nta procedure according to the kit directions . the eluted proteins were harvested and subjected to sds - page and western immunoblotting analysis ( figure 4 ) . this analysis showed that both of the recombinant slp and lsp - t ( figure 4(a ) ) proteins and the hybridized protein bands ( figure 4(b ) ) appeared in the predicted positions . it has been previously shown that the 30k - family proteins in the silkworm display antiapoptotic activity , especially 30kc6 [ 3 , 8 , 14 , 15 ] . however , whether the newly predicted slp and lsp - t proteins inhibit cellular apoptosis remains undetermined . thus , to study the effects of slp and lsp - t on cellular apoptosis , increasing concentrations of h2o2 were applied to the cells to examine the effects of such exposure on bmn cellular apoptosis . based on time - dependent experiments ( data not shown ) , the viability and apoptosis of bmn cells were determined 24  h after a brief 10  min treatment in the presence of h2o2 . dose - dependent effects of h2o2 on bmn cellular activity ( figure 5(a ) ) and apoptosis were also determined ( figure 5(b ) ) . however , we did not see any further significant increase in cellular apoptosis of bmn cells following treatment with h2o2 a dose greater than 0.5  mm . based on these data , the following experiments were pursued using 0.5  mm h2o2 . in these experiments , cultured bmn cells were preincubated with purified recombinant slp or lsp - t for 24  h and then treated with 0.5  mm h2o2 for 10  min . cells were then additionally treated with slp or lsp - t for 24  h. purified 30kc6 protein was used as a positive control . we found a statistically significant difference in respect to cell viability ( figure 5(c ) ) and dna fragmentation ( figure 5(d ) ) between h2o2-treated cells and those cells treated with slp , lsp - t , and 30kc6 . taken together , these data indicated that both recombinant slp and lsp - t have similar biological activities to 30kc6 protein . they could enhance cell viability , decrease dna fragmentation , and inhibit cell apoptosis following treatment of bmn cells to h2o2 . the effects of recombinant slp and lsp - t on cellular viability and apoptosis were further evaluated in h2o2-treated huvec cells . in the first instance , h2o2 was shown to induce cellular apoptosis in the huvec cell model . the dose - dependent effects of h2o2 on huvec cellular activity and apoptosis indicated that h2o2 decreased cell viability and increased cellular apoptosis ( figures 6(a ) and 6(b ) ) . there was a statistically significant difference in respect to cell viability and cell apoptosis between huvec cells treated with 0.5  mm h2o2 and controls . however , there was no further significant decrease in cell viability or increase in cellular apoptosis following treatment with h2o2 at doses > 0.5  mm . therefore , from the time - response experiments ( data not shown ) and the data described above , the viability and apoptosis of huvec cells were evaluated 24  h after treatment with 0.5  mm h2o2 for 30  min . the effects of slp and lsp - t on cell viability and apoptosis in h2o2-treated huvec cells were explored in the following studies . we found that both recombinant slp and lsp - t significantly increased cellular viability of h2o2-treated huvec cells as determined by mtt analysis ( figure 6(c ) ) . both recombinant slp and lsp - t significantly decreased dna fragmentation of huvec cells that had been pretreated with h2o2 ( figure 6(d ) ) . collectively , these data indicated that both recombinant slp and lsp - t have similar biological activities to 30kc6 protein . it has been previously reported that several kinds of 30k - family proteins are found in the silkworm , and they mainly serve important roles in energy metabolism and may simultaneously inhibit cellular apoptosis [ 69 ] . to date , only 3 kinds of 30kd proteins encoded by 30kc6 , 30kc12p , and 30kc19g1 have been shown to provide energy and serve as antiapoptotic proteins in the silkworm [ 69 ] . therefore , we set out to identify new members of the 30k family of proteins . comparative homology analyses by a bioinformatics approach , demonstrated that both slp and lsp - t shared a high degree of amino acid sequence homology with the three reported 30k - family proteins and exhibited a conserved lipoprotein-11 domain . further analysis revealed that both slp and lsp - t had predicted tertiary structures that were similar to those of the reported 30k - family proteins . therefore , it was reasonable to hypothesize that both slp and lsp - t may belong to the 30k - family of proteins . this hypothesis further raised the possibility that both slp and lsp - t may demonstrate an antiapoptotic activity . to study the biological function of these two proteins , we used rt - pcr to obtain the cdna sequences of slp and lsp - t from the fat bodies of the silkworm with the specific aim of expressing and purifying both the slp and lsp - t proteins . the results of bioinformatics analysis showed that the cdnas contained the signal peptides , indicating that they might be expressed as secrete proteins . whether the signal peptides of slp and lsp - t proteins were cleaved or not after expression in bmn cells , further investigations will be needed . previous studies have demonstrated that several 30k - family proteins in the silkworm were highly effective at inhibiting cellular apoptosis in actinomycin - induced sf9 cells and chemical - treated hela cells [ 6 , 8 ] . our previous work also indicated that 30k proteins from insects can inhibit apoptosis in h2o2-stimulated huvecs [ 7 , 16 ] . therefore , whether slp and lsp - t exert similar antiapoptotic activities was tested in the present study . h2o2-induced apoptosis models of bmn and huvec cells were first designed and subsequently used to explore the effects of slp and lsp - t on cellular viability and apoptosis . our results demonstrated that both slp and lsp - t at a final concentration of 1.5  g / ml ( in bmn cells ) and at 4.0  g / ml ( in huvecs ) significantly enhanced cellular viability and decreased dna fragmentation in both bmn and huvec cells that had been pretreated with h2o2 . our preliminary experiments , results showed that higher concentration of slp and lsp - t showed no further protection effects on bmn and huvec cellular apoptosis . these data indicated that both slp and lsp - t possess important antiapoptotic activities . although several previous studies have demonstrated that silkworm 30k proteins were highly effective at inhibiting cellular apoptosis in insect and mammalian cells [ 6 , 8 ] the potential antiapoptotic mechanism is still not clear . kim et al . found that overexpression of 30kc6 could reduce caspase 3 activity in vitro study , but in vivo study results showed that 30kc6 protein did not inhibit caspase 3 activity indicating that the apoptosis inhibitory activity site of 30kc6 might exist in the upstream of caspase 3 . choi et al . found that 30k protein could inhibit the release of cytochrome c from the mitochondria into the cytoplasm in cho cells , thus preventing the activation of caspase 3 as well as its downstream substrates , thereby inhibiting the apoptosis of cho cell . in summary , both slp and lsp - t represent new members of the 30k family of proteins both in terms of their structure and potentially important functions on cellular viability and cell death . further investigation is needed to investigate the potential antiapoptotic mechanism of slp and lsp - t .
the 30k family of proteins is important in energy metabolism and may play a role in inhibiting cellular apoptosis in silkworms ( bombyx mori ) . 
 several 30k - family proteins have been identified . in this study , two new silkworm genes , 
 referred to as slp ( nm 001126256 ) and lsp - t ( nm 001043443 ) , were analyzed by a bioinformatics approach according to the sequences of 30k proteins previously reported in the silkworm . 
 both slp and lsp - t shared more than 41% amino acid sequence homology with the reported 30k proteins and displayed a conserved domain consistent with that of lipoprotein-11 . 
 additionally , the cdna sequences of both slp and lsp - t were obtained from the fat bodies of silkworm larvae by reverse transcription polymerase chain reaction . 
 both genes were expressed in bmn cells using the bac - to - bac system . 
 purified slp and lsp - t were added to cultured bmn and human umbilical vein endothelial cells ( huvec ) that were treated with h2o2 . 
 both slp and lsp - t significantly enhanced the viability and suppressed dna fragmentation in h2o2 treated bmn and huvec cells . 
 this study suggested that slp and lsp - t exhibit similar biological activities as their known 30k - protein counterparts and mediate an inhibitory effect against h2o2-induced apoptosis .