Biopanning And Preliminary Research The Character Of DNA Binding Related Protein Genes From Newborn Larvae Of Trichinella Spiralis

Trichinellosis is a global zoonotic parasitic disease caused by nematode. It is still prevalence in China. More than 150 mammals could be infected besides human. Trichinellosis could not only cost great expenses in animal industry but also threaten people's health. The multiplicity of Trichinella spiralis antigens and impossibility to be reproduced in vitro made great difficulty for immunodiagnosis and prevention. After infecting host. Trichinella spiralis muscle larvae is infected with host, experience four exuviations to develop into adults in 30h. Consequencely, it gave birth to newborn larvae in 96h and newborn larvae invaded into all skeletal muscle by lymph and blood. Finally, newborn larvae grew up infective muscle larvae, which could survival in animal and human body for long term. Muscle larvae take in host nutrition and create enormous harm to human and animal.After entering the muscle cells, the newborn larvaes induced the muscle cells to have a series of physiological, biochemistry changes and the structure reconstruction in its rapid growth process, which caused muscle cells to lose their original characteristic and to form the cyst, thus provided the larvaes the ideal environment for long-term survival, and protected the larvas. The formation of the factors above are related with signal transduction between parasites and hosts. By presumption, after the newborn larva invasion, some proteins and factors which were expressed by them induce muscle cells chromosome to generate the regulative function, which caused the massive collogen expression and formed the cyst. Extracting the related proteins which were espressed by newborn larvaes and binding with muscle cells chromosome DNA will be the key of study the Trichinella spiralis cyst formation mechanism and signal transduction between parasites and hosts. Looked from the existing biological technology, using the phage display technique to biopanning the above demonstrated related protein genes will become an effective method.The phage display technique takes the phage as a carrier, made protein or the multi-peptide genes inserted into outer covering protein gene areas of phages directionally, which caused the extraneous proteins or the multi-peptides through expressed fusion proteins with the phage outer covering proteins and displayed on the surface of the phages. The proteins or the multi-peptides which were displayed might maintain the relative independent spatial structures and the biological activities. And then biopanning the expression special protein or the multi-peptide phages used affinity enrichment. The T7 phage display system was the newest one which was developed by Novagen Corporation. It could display broad-spectrum proteins or multi-peptides. The expressed multi-peptides or proteins as fusion proteins which were displayed on the surface of the phages with package of membrane proteins of phage.The T7 phage display library from adult and newborn larvae of Trichinella spiralis was constructed successfully which was the first time in domestic and foreign. And using the biopanning method which did not yet reported in domestic and foreign biopanned the phage display library by mouse muscle chromosome DNA. And the related protein gene sequences which were biopanned were analysised and the protein structure was forecasted. This research laid the foundation of the Trichinella spiralis cyst formation mechanism, signal transduction between parasites and hosts, prevention and treatment trichinianosis, and even parasitic disease.Adult and newborn larvae of Trichinella spiralis were collected; Total RNA was extracted by Trizol reagents; Messenger RNA was isolated from total RNA by Oligotex Centrifugalization; Then mRNA was reverse transcribed into double-stranded cDNA, end modification, EcoRI/HindIII adaptors ligation, the dscDNA was disgested with HindIII and EcoRI; After fractionated by SPIN-400 Mini Column, the dscDNA was ligated into the T7Select 10-3b vector arms. After packaged in vitro and amplificated, the quality of the library was identited. The result was the primitive library capacity was 3×105pfu, recombinant ratio was 98%, the ranges of the inserts were 250-2000bp, and the title of amplification library was 3×1012 pfu/mL. The T7 phage display cDNA library from adult and newborn larvae of Trichinella spiralis was constructed successfully. Extracted mouse muscle chromosome DNA, and then diged DNA onto the nitrocellulose membrane. Eluted the phages on DNA with the phage elution buffer, after drying, crossing linking, blocking and washing. Tited, amplificated, and then entered the next round biopanning after tited again. Finally the phages binding on the DNA were stable through 4 turns biopanning. The quantity of the phages that was binded with muscle chromosome DNA reached 1×107, indicated that the biopaning was saturated, the major part of phages in the library after amplification could be interacted with DNA at this time. The analytic results were as follow: Two genes encoding protein was homologous with unknown protein of Trichinella spiralis, and hypothetical ORF 11.30 of Trichinella spiralis. Five genes ecoding protein was homologous with putative nudix hydrolyase of Trichinella pseudospiralis and SJCHGC05997 protein of Schistosoma japonicum. Two genes ecoding protein was homologous with hypothetical protein CBG23797 of Caenorhabditis briggsae. One gene ecoding protein was homologous with TGF-beta-like ligand precursor of Trichinella spiralis. And three genes ecoding proteins were homologous with three different hypothetical protein of malaria parasite.After analyzed four genes could be used for researching the Trichinella spiralis cyst formation mechanism and signal transduction between parasites and hosts after the genes were analyzed. But the function of them need to further research. The conclusion was that we had established a technique successfully which was used nitrocellulose membrane to biopan phage display library with DNA as the foundation, and succeeded biopanning Trichinella spiralis newborn larva DNA binding related protein genes.This research was successfully constructed the T7 phage display library from adult and newborn larvae of Trichinella spiralis, and the biopaning technique had not reported in domestic and foreign yet. The phage display library had carried on biopanning by using mouse muscle chromosome DNA. Four genes could be used for researching the Trichinella spiralis cyst formation mechanism and signal transduction between parasites and hosts after the genes were analyzed. This research laid the foundation of the Trichinella spiralis cyst formation mechanism, signal transduction between parasites and hosts, prevention and treatment trichinianosis, and even parasitic disease.