Development Of FRET Methods For Lipopolysaccharide Detection

Lipopolysaccharide（LPS）is the component in gram-negative bacteria,known as exogenous"pyrogens",which can cause fever,microcirculation disorder,endotoxin shock and disseminated intravascular coagulation.Traditional detection method of limulus test e have the defects of false positives and cause the environmental damage becsuse of slaughtering horseshoe crabs Therefore,it is necessary to develop LPS methods with high sensitivity and specificity and environment-friend.In this study,endogenous proteins or peptides with high affinity to LPS were selected to construct the sensing unit,and the fluorescent proteins couple of m Turquoise/m Neon Green was used as the reporting unit.FRET protein sensor was constructed to realize the rapid detection of LPS with high sensitivity and specificity.This study mainly includes the following four aspects:A FRET biosensor for LPS detection has been constructed based on LPS-binding peptide（LBP）.In this study,the genes of the FRET donor m Turquoise and the FRET acceptor m Neon Green were fused with the gene of LBP to contruct the recombinant plasmids of p PIC9K-LBP-m Neon Green and p PIC9K-LBP-m Turquoise,respectively.The recombinant plasmids were transformed into Pichia pastoris GS115 and the recombinant proteins LBP-m Turquoise and LBP-m Neon Green were expressed and their biochemical and spectral properties were investigated.The FRET biosensor based on the LBP has been investigated to detect LPS.Firstly,The ratio of the protein concentration of LBP-m Turquoise and LBP-m Neon Green was optimized.Then,the FRET biosensor was used to detect LPS with linear relationship of 10 ng/m L to 200 ng/m L(Y=0.09834 log C_LPS+0.08543R²=0.99)and the detection limit of 2.03 ng/m L.The selectivity of the FRET biosensor for LPS was investigated by the fluorescence intensity response in the presence of various competing biological molecules.Finally,the FRET biosensor was used to detect the LPS in real water samples from Nanhu lake and tap.The results were compared with the limulus test with good reliability.Furthermore,the LBP-based FRET biosensor can make a distinction between Gram-negative and Gram-positive bacteria and fungus.2.A FRET biosensor for LPS detection has been constructed based on human Toll4 receptor（TLR4）and myeloid differentiation protein 2（MD2）.The design was based on the the characteristic that LPS can specifically bind and induce MD2-TLR4to form a dimer.The gene sequences of FRET donor m Turquoise and acceptor m Neon Green were fused with the gene sequences of MD2 and TLR4 To contruct the recombinantplasmidsPPIC9K-MD2-Toll4-m Turquoiseand PPIC9K-MD2-Toll4-mneongreen.The recombinants plasmids were transformed into Pichia pastoris GS115.The recombinant proteins MD2-TLR4-m Turquoise and MD2-TLR4-m Neon Green were expressed in Pichia pastoris and their biochemical and spectral properties were investigated.The FRET biosensor based on MD2-TLR4 has been used to detect LPS.Firstly,The ratio of the protein concentration of MD2-TLR4-m Turquoise and MD2-TLR4-m Neon Green was optimized,and then the FRET biosensor was used to detect LPS with linear relationship of 10 ng/m L-100 ng/m L(Y=0.01625 log C_LPS+0.19118 R²=0.98)and detection limit of 3.24 ng/m L.The selectivity of the FRET biosensor for LPS was investigated by the fluorescence intensity response in the presence of various competing biological molecules.Finally,the FRET biosensor was used to detect the LPS in real water samples from Nanhu lake and tap.The results were compared with the limulus test with good reliability.Furthermore,the LBP-based FRET biosensor can make a distinction between Gram-negative and Gram-positive bacteria and fungus.